Liposomal compositions of epoxyketone-based proteasome inhibitors

ABSTRACT

Liposomal compositions comprising peptide epoxyketone compounds are described, as well as methods of making and using such liposomal compositions. These liposomal compositions provide prolonged drug exposure (relative to non-liposomal compositions comprising peptide epoxyketone compounds) without significantly affecting biodistribution of the drug. Tolerability of peptide epoxyketone compounds was also enhanced when formulated in the liposomal compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/673,017, filed 18 Jul. 2012, now pending, and U.S. ProvisionalApplication Ser. No. 61/794,603, filed 15 Mar. 2013, now pending, bothof which applications are herein incorporated by reference in theirentireties.

TECHNICAL FIELD

The present invention relates to compositions for the treatment ofcancer, as well as methods of preparing such compositions. Aspects ofthe present invention include, but are not limited to, methods forformulating stable liposomal compositions comprising peptide epoxyketonecompounds, liposomal compositions comprising peptide epoxyketonecompounds, and methods of using such liposomal compositions.

BACKGROUND OF THE INVENTION

In eukaryotes, protein degradation is predominately mediated through theubiquitin pathway in which proteins targeted for destruction are ligatedto the 76 amino acid polypeptide ubiquitin. Once targeted, ubiquitinatedproteins then serve as substrates for the 26S proteasome, amulticatalytic protease, which cleaves proteins into short peptidesthrough the action of its three major proteolytic activities. Whilehaving a general function in intracellular protein turnover,proteasome-mediated degradation also plays a key role in many processessuch as major histocompatibility complex (MHC) class I presentation,apoptosis, cell growth regulation, NF-κB activation, antigen processing,and transduction of pro-inflammatory signals.

The 20S proteasome is a 700 kDa cylindrical-shaped multicatalyticprotease complex comprised of 28 subunits organized into four rings. Inyeast and other eukaryotes, 7 different a subunits form the outer ringsand 7 different β subunits comprise the inner rings. The a subunitsserve as binding sites for the 19S (PA700) and 11S (PA28) regulatorycomplexes, as well as a physical barrier for the inner proteolyticchamber formed by the two β subunit rings. Thus, in vivo, the proteasomeis believed to exist as a 26S particle (“the 26S proteasome”). In vivoexperiments have shown that inhibition of the 20S form of the proteasomecan be readily correlated to inhibition of 26S proteasome. Cleavage ofamino-terminal prosequences of β subunits during particle formationexposes amino-terminal threonine residues, which serve as the catalyticnucleophiles.

The subunits responsible for catalytic activity in proteasomes thuspossess an amino terminal nucleophilic residue, and these subunitsbelong to the family of N-terminal nucleophile (Ntn) hydrolases (wherethe nucleophilic N-terminal residue is, for example, Cys, Ser, Thr, orother nucleophilic moieties). This family includes, for example,penicillin G acylase (PGA), penicillin V acylase (PVA), glutamine PRPPamidotransferase (GAT), and bacterial glycosylasparaginase. In additionto the ubiquitously expressed β subunits, higher vertebrates alsopossess three interferon-γ-inducible β subunits (LMP7, LMP2 and MECL1),which replace their normal counterparts, β5, β1 and β2 respectively,thus altering the catalytic activities of the proteasome.

Through the use of different peptide substrates, three major proteolyticactivities have been defined for the eukaryote 20S proteasome:chymotrypsin-like activity (CT-L), which cleaves after large hydrophobicresidues; trypsin-like activity (T-L), which cleaves after basicresidues; and caspase-like (C-L), which cleaves after acidic residues.The major proteasome proteolytic activities appear to be contributed bydifferent catalytic sites, because inhibitors, point mutations in βsubunits and the exchange of interferon-γ-inducing β subunits alterthese activities to various degrees.

There are several examples of small molecules that have been used toinhibit proteasome activity and have been shown to be effective againstcancer, particularly multiple myeloma. However, these compoundsgenerally lack the specificity, stability, or potency necessary toexplore and exploit the roles of the proteasome at the cellular andmolecular level, and thus maximize their anti-cancer activity.

SUMMARY OF THE INVENTION

The present invention generally relates to liposomal compositions forincreasing the therapeutic activity of proteasome inhibitors,particularly by enhancing their pharmacokinetic properties. Aspects ofthe present invention include, liposomal compositions comprising apeptide epoxyketone compound, methods of making such compositions, andmethods of using such compositions.

In one aspect, the present invention relates to pharmaceutical liposomalcompositions. In some embodiments, the pharmaceutical liposomalcompositions comprise liposome entrapped peptide epoxyketone compound,wherein (i) liposomes of the liposomal composition comprise one or morelipids (e.g., L-α-phosphatidylcholine,1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and/or sphingomyelin),and in the liposomes the weight ratio of peptide epoxyketonecompound:lipid is between about 0.01:1 and about 1:1. In a preferredembodiment, the weight ratio of peptide epoxyketone compound:lipid isbetween about 0.05:1 to about 0.5:1. Typically, the liposomes have anaverage size of between about 0.05 microns to about 0.5 microns,preferably an average size of between about 0.05 microns to about 0.2microns.

Embodiments of the pharmaceutical liposomal compositions presentinvention include, but are not limited to, the lipids of the liposomescomprising a phospholipid, a cholesterol, a hydrophilicpolymer-derivatized lipid, and/or combinations thereof.

In some embodiments of this aspect of the present invention, theliposomal composition comprises liposomes comprising the peptideepoxyketone compound and a solubilizing agent (e.g., a compound).

The pharmaceutical liposomal composition of the present invention canalso include one or more excipients.

In other aspects, the present invention relates to methods of making thepharmaceutical liposomal compositions described herein. One method ofmaking a pharmaceutical liposomal composition comprises preparing adried film comprising a peptide epoxyketone compound and at least onelipid, and rehydrating the dried film with an aqueous solution to form aliposomal composition comprising liposomes and an aqueous solution.Another method of making a pharmaceutical liposomal compositioncomprises preparing a dried film comprising one or more lipids andrehydrating the dried film with an aqueous solution comprising a peptideepoxyketone compound to form a liposomal composition comprisingliposomes dispersed in aqueous solution. Typically the aqueous solutioncomprises a peptide epoxyketone compound and a solubilizing agent. Yetanother method of making a pharmaceutical liposomal compositioncomprises preparing a lipid solution comprising one or more lipids and asolvent and injecting the lipid solution into an aqueous solutioncomprising a peptide epoxyketone compound. Typically the aqueoussolution comprises a peptide epoxyketone compound and a solubilizingagent.

In preferred embodiments, the methods of making pharmaceutical liposomalcompositions further comprises sizing the liposomes to have an averagesize of between about 0.05 microns to about 0.5 microns (preferably anaverage size of between about 0.05 microns to about 0.2 microns).

In some embodiments, excess peptide epoxyketone compounds are removedfrom the non-encapsulated aqueous solution.

In some embodiments, the method further comprises adding one or moreexcipients, including, but not limited to, a pH adjusting agent (e.g., abuffer) and/or an agent to maintain isotonicity, to the aqueous solutionin which the liposomes are dispersed.

In another aspect, the present invention relates to pharmaceuticalliposomal compositions made by the methods of the invention.

In a further aspect, the present invention relates to methods oftreating a disease or condition in a subject in need of treatment,comprising administering a therapeutically effective amount of apharmaceutical liposomal composition comprising liposomes comprising apeptide epoxyketone compound. In some embodiments the methods oftreating further comprise simultaneous, sequential, or separateadministration of a therapeutically effective amount of anothertherapeutic agent, for example, a chemotherapeutic agent, a cytokine, asteroid, an immunotherapeutic agent, or combinations thereof.

In another aspect the present invention relates to dry pharmaceuticalcompositions comprising, one or more amphipathic lipids (e.g., aphospholipid), and a peptide epoxyketone compound.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents the characterization of exemplary liposomal carfilzomibcompositions (A-G) used to generate the data presented in the Examplesand Tables herein.

FIG. 2A presents data related to pharmacodynamic responses in BALB/Cmice to different compositions of carfilzomib. In the figure, thevertical axis is the percent (%) enzymatic activity relative to vehicle,wherein the enzymatic activity corresponds to proteasome CT-L activityin whole blood (primarily erythrocytes). Three groups of data arepresented on the horizontal axis as follows. The first group,represented in the figure as cross-hatched bars, presents data for aninjectable composition of carfilzomib (CFZ) formulated in 10%sulfobutylether beta cyclodextrin (SBE-β-CD, also referred to herein asSBE-CD), and 10 mM Citrate, pH 3.5 (non-liposomal): the first barpresents control data for the placebo vehicle without carfilzomib, thesecond bar presents data for CFZ SBE-CD at 1 hour, the third barpresents data for CFZ SBE-CD at 8 hours, and the fourth bar presentsdata for CFZ SBE-CD at 24 hours. The second group, represented in thefigure as diagonal-lined bars, presents data for a liposomal compositionof carfilzomib: the first bar presents control data for the liposomalvehicle without carfilzomib, the second bar presents data for thecarfilzomib liposomal composition at 1 hour, the third bar presents datafor the carfilzomib liposomal composition at 8 hours, and the fourth barpresents data for the carfilzomib liposomal composition at 24 hours. Thethird group, represented in the figure as white, outlined bars, presentsdata for a pegylated liposomal composition of carfilzomib: the first barpresents control data for the pegylated liposomal vehicle withoutcarfilzomib, the second bar presents data for the carfilzomib pegylatedliposomal composition at 1 hour, the third bar presents data for thecarfilzomib pegylated liposomal composition at 8 hours, and the fourthbar presents data for the carfilzomib pegylated liposomal composition at24 hours.

FIG. 2B presents data related to pharmacodynamic responses in BALB/Cmice to different compositions of carfilzomib. In the figure, thevertical axis is the percent (%) enzymatic activity relative to vehicle,wherein the enzymatic activity corresponds to proteasome CT-L activityin adrenal tissue. Three groups of data are presented on the horizontalaxis as follows. The first group, represented in the figure ascross-hatched bars, presents data for injectable CFZ SBE-CD(non-liposomal): the first bar presents control data for the vehiclewithout carfilzomib, the second bar presents data for CFZ SBE-CD at 1hour, the third bar presents data for CFZ SBE-CD at 8 hours, and thefourth bar presents data for CFZ SBE-CD at 24 hours. The second group,represented in the figure as diagonal-lined bars, presents data for aliposomal composition of carfilzomib: the first bar presents controldata for the liposomal vehicle without carfilzomib, the second barpresents data for the carfilzomib liposomal composition at 1 hour, thethird bar presents data for the carfilzomib liposomal composition at 8hours, and the fourth bar presents data for the carfilzomib liposomalcomposition at 24 hours. The third group, represented in the figure aswhite, outlined bars, presents data for a pegylated liposomalcomposition of carfilzomib: the first bar presents control data for thepegylated liposomal vehicle without carfilzomib, the second bar presentsdata for the carfilzomib pegylated liposomal composition at 1 hour, thethird bar presents data for the carfilzomib pegylated liposomalcomposition at 8 hours, and the fourth bar presents data for thecarfilzomib pegylated liposomal composition at 24 hours.

FIG. 2C presents data related to pharmacodynamic responses in BALB/Cmice to different compositions of carfilzomib. In the figure, thevertical axis is the percent (%) enzymatic activity relative to vehicle,wherein the enzymatic activity corresponds to proteasome CT-L activityin liver tissue. Three groups of data are presented on the horizontalaxis as follows. The first group, represented in the figure ascross-hatched bars, presents data for injectable CFZ SBE-CD(non-liposomal): the first bar presents control data for the vehiclewithout carfilzomib, the second bar presents data for the carfilzomibSBE-CD composition at 1 hour, the third bar presents data for thecarfilzomib composition at 8 hours, and the fourth bar presents data forthe carfilzomib composition at 24 hours. The second group, representedin the figure as diagonal-lined bars, presents data for a liposomalcomposition of carfilzomib: the first bar presents control data for theliposomal vehicle without carfilzomib, the second bar presents data forthe carfilzomib liposomal composition at 1 hour, the third bar presentsdata for the carfilzomib liposomal composition at 8 hours, and thefourth bar presents data for the carfilzomib liposomal composition at 24hours. The third group, represented in the figure as white, outlinedbars, presents data for a pegylated liposomal composition ofcarfilzomib: the first bar presents control data for the pegylatedliposomal vehicle without carfilzomib, the second bar presents data forthe carfilzomib pegylated liposomal composition at 1 hour, the third barpresents data for the carfilzomib pegylated liposomal composition at 8hours, and the fourth bar presents data for the carfilzomib pegylatedliposomal composition at 24 hours.

FIG. 2D presents data related to pharmacodynamic responses in BALB/Cmice to different compositions of carfilzomib. In the figure, thevertical axis is the percent (%) enzymatic activity relative to vehicle,wherein the enzymatic activity corresponds to proteasome CT-L activityin heart tissue. Three groups of data are presented on the horizontalaxis as follows. The first group, represented in the figure ascross-hatched bars, presents data for injectable CFZ SBE-CD(non-liposomal): the first bar presents control data for the vehiclewithout carfilzomib, the second bar presents data for CFZ SBE-CD at 1hour, the third bar presents data for CFZ SBE-CD at 8 hours, and thefourth bar presents data for CFZ SBE-CD at 24 hours. The second group,represented in the figure as diagonal-lined bars, presents data for aliposomal composition of carfilzomib: the first bar presents controldata for the liposomal vehicle without carfilzomib, the second barpresents data for the carfilzomib liposomal composition at 1 hour, thethird bar presents data for the carfilzomib liposomal composition at 8hours, and the fourth bar presents data for the carfilzomib liposomalcomposition at 24 hours. The third group, represented in the figure aswhite, outlined bars, presents data for a pegylated liposomalcomposition of carfilzomib: the first bar presents control data for thepegylated liposomal vehicle without carfilzomib, the second bar presentsdata for the carfilzomib pegylated liposomal composition at 1 hour, thethird bar presents data for the carfilzomib pegylated liposomalcomposition at 8 hours, and the fourth bar presents data for thecarfilzomib pegylated liposomal composition at 24 hours.

FIG. 3A presents data related to pharmacodynamic responses in BALB/Cmice to different compositions of carfilzomib. In the figure, thevertical axis (CT-L activity) is the percent (%) enzymatic activityrelative to a corresponding vehicle without carfilzomib (CFZ), whereinthe enzymatic activity corresponds to proteasome CT-L activity in wholeblood (primarily erythrocytes). The horizontal axis is the time in hours(Hour). Four groups of data are presented. The first group (open squarescontaining an X) presents data for an injectable composition ofcarfilzomib (CFZ) formulated in 10% sulfobutylether beta cyclodextrin(SBE-CD), and 10 mM Citrate, pH 3.5, (non-liposomal) administered at 5mg/kg with data points at 0, 1, 4, 6, 8, and 24 hours. The second group(open circles containing an X) presents data for an injectablecomposition of carfilzomib (CFZ) formulated in 10% sulfobutylether betacyclodextrin (SBE-CD), and 10 mM Citrate, pH 3.5, (non-liposomal)administered at 10 mg/kg with data points at 0, 1, 8, and 24 hours. Thethird group (solid squares) presents data for a pegylated liposomalcomposition of carfilzomib wherein the aqueous core of the pegylatedliposomes comprises carfilzomib and SBE-CD (ap-L11) administered at 5mg/kg with data points at 0, 1, 4, 6, and 24 hours. The fourth group(solid circles) presents data for a pegylated liposomal composition ofcarfilzomib wherein the aqueous core of the pegylated liposomescomprises carfilzomib and SBE-CD (ap-L11) administered at 15 mg/kg withdata points at 0, 1, 4, 6, and 24 hours.

FIG. 3B presents data related to pharmacodynamic responses in BALB/Cmice to different compositions of carfilzomib. In the figure, thevertical axis (CT-L activity) is the percent (%) enzymatic activityrelative to a corresponding vehicle without carfilzomib (CFZ), whereinthe enzymatic activity corresponds to proteasome CT-L activity in hearttissue. The horizontal axis is the time in hours (Hour). Four groups ofdata are presented. The first group (open squares containing an X)presents data for an injectable composition of carfilzomib (CFZ)formulated in 10% sulfobutylether beta cyclodextrin (SBE-CD), and 10 mMCitrate, pH 3.5, (non-liposomal) administered at 5 mg/kg with datapoints at 0, 1, 4, 6, 8, and 24 hours. The second group (open circlescontaining an X) presents data for an injectable composition ofcarfilzomib (CFZ) formulated in 10% sulfobutylether beta cyclodextrin(SBE-CD), and 10 mM Citrate, pH 3.5, (non-liposomal) administered at 10mg/kg with data points at 0, 1, 8, and 24 hours. The third group (solidsquares) presents data for a pegylated liposomal composition ofcarfilzomib wherein the aqueous core of the pegylated liposomescomprises carfilzomib and SBE-CD (ap-L11) administered at 5 mg/kg withdata points at 0, 1, 4, 6, and 24 hours. The fourth group (solidcircles) presents data for a pegylated liposomal composition ofcarfilzomib wherein the aqueous core of the pegylated liposomescomprises carfilzomib and SBE-CD (ap-L11) administered at 15 mg/kg withdata points at 0, 1, 4, 6, and 24 hours.

FIG. 3C presents data related to pharmacodynamic responses in BALB/Cmice to different compositions of carfilzomib. In the figure, thevertical axis (CT-L activity) is the percent (%) enzymatic activityrelative to a corresponding vehicle without carfilzomib (CFZ), whereinthe enzymatic activity corresponds to proteasome CT-L activity in livertissue. The horizontal axis is the time in hours (Hour). Four groups ofdata are presented. The first group (open squares containing an X)presents data for an injectable composition of carfilzomib (CFZ)formulated in 10% sulfobutylether beta cyclodextrin (SBE-CD), and 10 mMCitrate, pH 3.5, (non-liposomal) administered at 5 mg/kg with datapoints at 0, 1, 4, 6, 8, and 24 hours. The second group (open circlescontaining an X) presents data for an injectable composition ofcarfilzomib (CFZ) formulated in 10% sulfobutylether beta cyclodextrin(SBE-CD), and 10 mM Citrate, pH 3.5, (non-liposomal) administered at 10mg/kg with data points at 0, 1, 8, and 24 hours. The third group (solidsquares) presents data for a pegylated liposomal composition ofcarfilzomib wherein the aqueous core of the pegylated liposomescomprises carfilzomib and SBE-CD (ap-L11) administered at 5 mg/kg withdata points at 0, 1, 4, 6, and 24 hours. The fourth group (solidcircles) presents data for a pegylated liposomal composition ofcarfilzomib wherein the aqueous core of the pegylated liposomescomprises carfilzomib and SBE-CD (ap-L11) administered at 15 mg/kg withdata points at 0, 1, 4, 6, and 24 hours.

FIG. 3D presents data related to pharmacodynamic responses in BALB/Cmice to different compositions of carfilzomib. In the figure, thevertical axis (CT-L activity) is the percent (%) enzymatic activityrelative to a corresponding vehicle without carfilzomib (CFZ), whereinthe enzymatic activity corresponds to proteasome CT-L activity inadrenal tissue. The horizontal axis is the time in hours (Hour). Fourgroups of data are presented. The first group (open squares containingan X) presents data for an injectable composition of carfilzomib (CFZ)formulated in 10% sulfobutylether beta cyclodextrin (SBE-CD), and 10 mMCitrate, pH 3.5, (non-liposomal) administered at 5 mg/kg with datapoints at 0, 1, 4, 6, 8, and 24 hours. The second group (open circlescontaining an X) presents data for an injectable composition ofcarfilzomib (CFZ) formulated in 10% sulfobutylether beta cyclodextrin(SBE-CD), and 10 mM Citrate, pH 3.5, (non-liposomal) administered at 10mg/kg with data points at 0, 1, 8, and 24 hours. The third group (solidsquares) presents data for a pegylated liposomal composition ofcarfilzomib wherein the aqueous core of the pegylated liposomescomprises carfilzomib and SBE-CD (ap-L11) administered at 5 mg/kg withdata points at 0, 1, 4, 6, and 24 hours. The fourth group (solidcircles) presents data for a pegylated liposomal composition ofcarfilzomib wherein the aqueous core of the pegylated liposomescomprises carfilzomib and SBE-CD (ap-L11) administered at 15 mg/kg withdata points at 0, 1, 4, 6, and 24 hours.

FIG. 4 presents data related to the circulation half-life in BALB/C miceof different compositions of carfilzomib. In the figure, the verticalaxis is the concentration of carfilzomib in umol/L (Concentration(umol/L)), and the horizontal axis is the time in minutes (Time (min)).The line with open squares containing an X corresponds to administrationof 5 mg/kg of an injectable carfilzomib SBE-CD composition(non-liposomal). The line with solid squares corresponds toadministration of 5 mg/kg of apL11, a pegylated liposomal composition ofcarfilzomib wherein the aqueous core of the pegylated liposomescomprises carfilzomib and SBE-CD. The line with solid circlescorresponds to administration of 15 mg/kg of apL11, a pegylatedliposomal composition of carfilzomib wherein the aqueous core of thepegylated liposomes comprises carfilzomib and SBE-CD.

FIG. 5A presents data related to pharmacodynamic responses in BALB/Cmice to different compositions of carfilzomib. In the figure, thevertical axis (CT-L Activity) is the percent (%) enzymatic activityrelative to a corresponding vehicle without carfilzomib (CFZ), whereinthe enzymatic activity corresponds to proteasome CT-L activity in wholeblood (primarily erythrocytes). The horizontal axis is the time in hours(Hour). Two groups of data are presented. The first group (open circles)presents data for an injectable composition of carfilzomib (CFZ)formulated in 10% sulfobutylether beta cyclodextrin (SBE-CD), and 10 mMCitrate, pH 3.5, (non-liposomal) administered at 10 mg/kg with datapoints at 0, 1, 8, and 24 hours. The second group (solid squares)presents data for a liposomal composition of carfilzomib comprisingliposomes comprising entrapped carfilzomib (pL-6) administered at 15mg/kg with data points at 0, 1, 4, 6, and 24 hours.

FIG. 5B presents data related to pharmacodynamic responses in BALB/Cmice to different compositions of carfilzomib. In the figure, thevertical axis (CT-L Activity) is the percent (%) enzymatic activityrelative to a corresponding vehicle without carfilzomib (CFZ), whereinthe enzymatic activity corresponds to proteasome CT-L activity in hearttissue. The horizontal axis is the time in hours (Hour). Two groups ofdata are presented. The first group (open circles) presents data for aninjectable composition of carfilzomib (CFZ) formulated in 10%sulfobutylether beta cyclodextrin (SBE-CD), and 10 mM Citrate, pH 3.5,(non-liposomal) administered at 10 mg/kg with data points at 0, 1, 8,and 24 hours. The second group (solid squares) presents data for aliposomal composition of carfilzomib comprising liposomes comprisingentrapped carfilzomib (pL-6) administered at 15 mg/kg with data pointsat 0, 1, 4, 6, and 24 hours.

FIG. 5C presents data related to pharmacodynamic responses in BALB/Cmice to different compositions of carfilzomib. In the figure, thevertical axis (CT-L Activity) is the percent (%) enzymatic activityrelative to a corresponding vehicle without carfilzomib (CFZ), whereinthe enzymatic activity corresponds to proteasome CT-L activity in livertissue. The horizontal axis is the time in hours (Hour). Two groups ofdata are presented. The first group (open circles) presents data for aninjectable composition of carfilzomib (CFZ) formulated in 10%sulfobutylether beta cyclodextrin (SBE-CD), and 10 mM Citrate, pH 3.5,(non-liposomal) administered at 10 mg/kg with data points at 0, 1, 8,and 24 hours. The second group (solid squares) presents data for aliposomal composition of carfilzomib comprising liposomes comprisingentrapped carfilzomib (pL-6) administered at 15 mg/kg with data pointsat 0, 1, 4, 6, and 24 hours.

FIG. 5D presents data related to pharmacodynamic responses in BALB/Cmice to different compositions of carfilzomib. In the figure, thevertical axis (CT-L Activity) is the percent (%) enzymatic activityrelative to a corresponding vehicle without carfilzomib (CFZ), whereinthe enzymatic activity corresponds to proteasome CT-L activity inadrenal tissue. The horizontal axis is the time in hours (Hour). Twogroups of data are presented. The first group (open circles) presentsdata for an injectable composition of carfilzomib (CFZ) formulated in10% sulfobutylether beta cyclodextrin (SBE-CD), and 10 mM Citrate, pH3.5, (non-liposomal) administered at 10 mg/kg with data points at 0, 1,8, and 24 hours. The second group (solid squares) presents data for aliposomal composition of carfilzomib comprising liposomes comprisingentrapped carfilzomib (pL-6) administered at 15 mg/kg with data pointsat 0, 1, 4, 6, and 24 hours.

FIG. 6 presents data related to the circulation half-life in BALB/C miceof different compositions of carfilzomib. In the figure, the verticalaxis is the concentration of carfilzomib in umol/L (Concentration(umol/L)), and the horizontal axis is the time post dose in minutes(Time Post Dose (min)). The line with open circles corresponds toadministration of 5 mg/kg of an injectable carfilzomib SBE-CDcomposition (non-liposomal). The line with solid squares corresponds toadministration of 15 mg/kg of a liposomal composition of carfilzomibcomprising liposomes comprising entrapped carfilzomib (pL-6).

FIG. 7 presents data related to the dosing frequency of differentcompositions of carfilzomib. In the figure, the vertical axis is tumorvolume in mm³ (Tumor Volume (mm³)), and the horizontal axis is dayspost-tumor challenge (Days). The line (top line in the figure at 30days) with open circles correspond to once-weekly administration ofvehicle (liposomes comprising 12.5 mg/mL EPC, 3.1 mg/mL Cholesterol, 1.2mg/mL mPEG-DSPE with no carfilzomib); the line with open squarescorresponds to 5 mg/kg of an injectable carfilzomib SBE-CD composition(non-liposomal) administered on days 1 and 2 of each week; the line withsolid circles corresponds to administration of pL6=2 mg/mL CFZ, 12.5mg/mL Sphingomylin, 3.2 mg/mL cholesterol, 1.3 mg/mL mPEG-DSPE providinga dose of 10 mg/kg of carfilzomib administered on days 1 and 2 of eachweek. The line with open triangles corresponds to administration ofpL6=2 mg/mL CFZ, 12.5 mg/mL Sphingomylin, 3.2 mg/mL cholesterol, 1.3mg/mL mPEG-DSPE providing a dose of 15 mg/kg of carfilzomib administeredonce weekly. The solid arrow indicates the start of the dosing period.

DETAILED DESCRIPTION OF THE INVENTION

All patents, publications, and patent applications cited in thisspecification are herein incorporated by reference as if each individualpatent, publication, or patent application was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes.

1.0.0 Definitions

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. As used in this specification and the appended claims,the singular forms “a,” “an” and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a lipid” includes one or more lipids, or mixtures of lipids, referenceto “a hydrophilic polymer” includes one or more hydrophilic polymers, ormixtures of hydrophilic polymers, reference to “a drug” includes one ormore drugs, and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although other methods andmaterials similar, or equivalent, to those described herein can be usedin the practice of the present invention, the preferred materials andmethods are described herein.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

The term “enzyme” as used herein refers to any partially or whollyproteinaceous molecule that carries out a chemical reaction in acatalytic manner. Such enzymes can be native enzymes, fusion enzymes,proenzymes, apoenzymes, denatured enzymes, farnesylated enzymes,ubiquitinated enzymes, fatty acylated enzymes, gerangeranylated enzymes,GPI-linked enzymes, lipid-linked enzymes, prenylated enzymes,naturally-occurring or artificially generated mutant enzymes, enzymeswith side chain or backbone modifications, enzymes having leadersequences, and enzymes complexed with non-proteinaceous material, suchas proteoglycans and proteoliposomes. Enzymes can be made by any means,including natural expression, promoted expression, cloning, varioussolution-based and solid-based peptide syntheses, and similar methodsknown to those skilled in the art.

The term “C_(x-y) alkyl” as used herein refers to substituted orunsubstituted saturated hydrocarbon groups, including straight-chainalkyl and branched-chain alkyl groups that contain from x to y carbonsin the chain, including haloalkyl groups such as trifluoromethyl and2,2,2-trifluoroethyl, etc. C₀ alkyl indicates a hydrogen where the groupis in a terminal position, a bond if internal. The terms “C_(2-y)alkenyl” and “C_(2-y) alkynyl” refer to substituted or unsubstitutedunsaturated aliphatic groups analogous in length and possiblesubstitution to the alkyls, but that contain at least one double ortriple bond respectively.

The term “alkoxy” as used herein refers to an alkyl group having anoxygen attached thereto. Representative alkoxy groups include methoxy,ethoxy, propoxy, tert-butoxy, and the like.

The term “ether” as used herein refers to two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxy.

The term “C₁₋₆ alkoxyalkyl” as used herein refers to a C₁₋₆ alkyl groupsubstituted with an alkoxy group, thereby forming an ether.

The term “C₁₋₆ aralkyl” as used herein refers to a C₁₋₆ alkyl groupsubstituted with an aryl group.

The terms “amine” and “amino” as used herein are art-recognized andrefer to both unsubstituted and substituted amines and salts thereof,e.g., a moiety that can be represented by the general formulae:

wherein R⁹, R¹⁰ and R^(10′) each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R⁸, or R⁹ and R¹⁰ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R⁸ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocyclyl or a polycyclyl; and m is zero or aninteger from 1 to 8. In preferred embodiments, only one of R⁹ or R¹⁰ canbe a carbonyl, e.g., R⁹, R¹⁰, and the nitrogen together do not form animide. In even more preferred embodiments, R⁹ and R¹⁰ (and optionallyR^(10′)) each independently represent a hydrogen, an alkyl, an alkenyl,or —(CH₂)_(m)—R⁸. In certain embodiments, the amino group is basic,meaning the protonated form has a pK_(a)≧7.00.

The terms “amide” and “amido” are art-recognized as referring to anamino-substituted carbonyl and including a moiety that can berepresented by the general formula:

wherein R⁹, R¹⁰ are as defined above. Preferred embodiments of the amidedoes not include imides that can be unstable.

The term “aryl” as used herein refers to 5-membered, 6-membered, and7-membered substituted or unsubstituted single-ring aromatic groups inwhich each atom of the ring is carbon. The term “aryl” also includespolycyclic ring systems having two or more cyclic rings in which two ormore carbons are common to two adjoining rings, wherein at least one ofthe rings is aromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline,and the like.

The terms “carbocycle” and “carbocyclyl” as used herein refer to anon-aromatic substituted or unsubstituted ring in which each atom of thering is carbon. The terms “carbocycle” and “carbocyclyl” also includepolycyclic ring systems having two or more cyclic rings in which two ormore carbons are common to two adjoining rings wherein at least one ofthe rings is carbocyclic, e.g., the other cyclic rings can becycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls.

The term “carbonyl” as used herein is art-recognized and refers tomoieties as can be represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R″represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R⁸, or apharmaceutically acceptable salt; R^(11′) represents a hydrogen, analkyl, an alkenyl, or —(CH₂)_(m)—R⁸, where m and R⁸ are as definedbelow. Where X is an oxygen and R¹¹ or R^(11′) is not hydrogen, theformula represents an “ester.” Where X is an oxygen and R¹¹ is ahydrogen, the formula represents a “carboxylic acid.”

The term “C₁₋₆ heteroaralkyl” as used herein refers to a C₁₋₆ alkylgroup substituted with a heteroaryl group.

The term “heteroaryl” as used herein refers to substituted orunsubstituted aromatic 5-membered to 7-membered ring structures, morepreferably 5-membered to 6-membered rings, whose ring structures includeone to four heteroatoms. The term “heteroaryl” also includes polycyclicring systems having two or more cyclic rings in which two or morecarbons are common to two adjoining rings, wherein at least one of therings is heteroaromatic, e.g., the other cyclic rings can becycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls. Heteroaryl groups include, for example, pyrrole, furan,thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine,pyrazine, pyridazine, pyrimidine, and the like.

The term “heteroatom” as used herein refers to an atom of any elementother than carbon or hydrogen. Preferred heteroatoms are nitrogen,oxygen, phosphorus, and sulfur.

The terms “heterocyclyl” and “heterocyclic group” as used herein referto substituted or unsubstituted non-aromatic 3-membered to 10-memberedring structures, more preferably 3-membered to 7-membered rings, whosering structures include one to four heteroatoms. The term terms“heterocyclyl” or “heterocyclic group” also include polycyclic ringsystems having two or more cyclic rings in which two or more carbons arecommon to two adjoining rings, wherein at least one of the rings isheterocyclic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Heterocyclyl groups include, for example, piperidine, piperazine,pyrrolidine, morpholine, lactones, lactams, and the like.

The term “C₁₋₆hydroxyalkyl” as used herein refers to a C₁₋₆alkyl groupsubstituted with a hydroxy group.

The term “thioether” as used herein refers to an alkyl group having asulfur moiety attached thereto. In preferred embodiments, the“thioether” is represented by —S-alkyl. Representative thioether groupsinclude methylthio, ethylthio, and the like.

The term “substituted” as used herein refers to moieties havingsubstituents replacing a hydrogen on one or more carbons of thebackbone. The terms “substitution” or “substituted with” include theimplicit proviso that such substitution is in accordance with permittedvalence of the substituted atom and the substituent, and that thesubstitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc. As used herein, the term “substituted” iscontemplated to include all permissible substituents of organiccompounds. In a broad aspect, the permissible substituents includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and non-aromatic substituents of organiccompounds. The permissible substituents can be one or more and the sameor different for appropriate organic compounds. For purposes of thisinvention, the heteroatoms such as nitrogen may have hydrogensubstituents and/or any permissible substituents of organic compoundsdescribed herein that satisfy the valences of the heteroatoms.Substituents can include, for example, a halogen, a hydroxyl, a carbonyl(such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), athiocarbonyl (such as a thioester, a thioacetate, or a thioformate), analkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, anamino, an amido, an amidine, an imine, a cyano, a nitro, an azido, asulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, asulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic orheteroaromatic moiety. It will be understood by those skilled in the artthat the moieties substituted on the hydrocarbon chain can themselves besubstituted, if appropriate.

The term “inhibitor” as used herein refers to a compound that blocks orreduces an activity of an enzyme (e g., inhibition of proteolyticcleavage of standard fluorogenic peptide substrates such assuc-LLVY-AMC, Box-LLR-AMC and Z-LLE-AMC, inhibition of various catalyticactivities of the 20S proteasome). An inhibitor can act withcompetitive, uncompetitive, or noncompetitive inhibition. An inhibitorcan bind reversibly or irreversibly, and therefore the term includescompounds that are suicide substrates of an enzyme. An inhibitor canmodify one or more sites on or near the active site of the enzyme, or itcan cause a conformational change elsewhere on the enzyme.

The term “peptide” as used herein refers not only to standard amidelinkage with standard α-substituents, but also to commonly usedpeptidomimetics, other modified linkages, non-naturally occurring sidechains, and side chain modifications, for example, as described in U.S.Pat. No. 7,417,042.

The terms “polycyclyl” and “polycyclic” as used herein refer to two ormore rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,heteroaryls, and/or heterocyclyls) in which two or more carbons arecommon to two adjoining rings, e.g., the rings are “fused rings.” Eachof the rings of the polycycle can be substituted or unsubstituted.

The term “prodrug” as used herein refers to compounds that, underphysiological conditions, are converted into therapeutically activeagents. A common method for making a prodrug is to include selectedmoieties that are hydrolyzed under physiological conditions to revealthe desired molecule. In other embodiments, the prodrug is converted byan enzymatic activity of the host animal.

The term “preventing” as used herein is art-recognized, and when used inrelation to a condition, such as a local recurrence (e.g., pain), adisease such as cancer, a syndrome complex such as heart failure or anyother medical condition, is well understood in the art, and includesadministration of a composition that reduces the frequency of, or delaysthe onset of, symptoms of a medical condition in a subject relative to asubject who does not receive the composition. Thus, prevention of cancerincludes, for example, reducing the number of detectable cancerousgrowths in a population of subjects receiving a prophylactic treatmentrelative to an untreated control population, and/or delaying theappearance of detectable cancerous growths in a treated populationversus an untreated control population, e.g., by a statistically and/orclinically significant amount. Prevention of an infection includes, forexample, reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

The term “prophylactic or therapeutic” treatment, as used herein, isart-recognized and refers to administration to the subject of one ormore of the subject compositions. If it is administered prior toclinical manifestation of the unwanted condition (e.g., disease or otherunwanted state of the subject) then the treatment is prophylactic,(i.e., it protects the subject against developing the unwantedcondition), whereas if it is administered after manifestation of theunwanted condition, the treatment is therapeutic, (i.e., it is intendedto diminish, ameliorate, or stabilize the existing unwanted condition orside effects thereof).

The term “proteasome” as used herein refers to immuno- and constitutiveproteasomes.

The term “therapeutically effective amount” as used herein refers to anamount of the compound(s) in a preparation that, when administered aspart of a desired dosage regimen (to a mammal, preferably a human)alleviates a symptom, ameliorates a condition, or slows the onset ofdisease conditions according to clinically acceptable standards for thedisorder or condition to be treated or the cosmetic purpose, e.g., at areasonable benefit/risk ratio applicable to any medical treatment.

The terms “treating” and “treatment” as used herein refer to reversing,reducing, or arresting the symptoms, clinical signs, and underlyingpathology of a condition in manner to improve or stabilize a subject'scondition.

The term “amphipathic lipids” as used herein refers to molecules thatare mostly lipid-like (hydrophobic) in structure, but at one end have aregion that is polar, charged, or a combination of polar and charged(hydrophilic). The hydrophilic region is referred to as the head group,and the lipid portion is known as the tail group(s). Examples ofamphipathic lipids include phospholipids, glycolipids, andsphingolipids.

The terms “hydrophilic polymer” and “water-soluble polymer” as usedherein refer to polymers, for example, polyethylene glycol (PEG) orother polyethoxylated polymers, used to shield liposomes and therebyenhance liposomal circulatory half-life. “Hydrophilic polymer”encompasses free hydrophilic polymers associated non-covalently with theliposomes and hydrophilic polymers that are conjugated or covalentlylinked to a component of the liposome (e.g., PEG-modified lipids).Additional exemplary hydrophilic polymers include, but are not limitedto, polyvinyl alcohol, polylactic acid, polyglycolic acid,polyacrylamide, polyvinylpyrrolidone, polyglycerol, polyaxozlines, etc.

The term “free sterol” as used herein refers to a sterol that is notcovalently bound to another compound. “Free cholesterol” refers tocholesterol that is not covalently bound as a moiety in asterol-modified amphiphilic lipid compound.

The terms “sterol” and “steroid alcohols” as used herein refer to thesubgroup of steroids having a free hydroxyl or a derivative thereof.Exemplary sterols include, but are not limited to, the class cholesteroland derivatives thereof, the class phytosterols and derivatives thereof,and the class fungal sterols and derivatives thereof. Sterols can benatural or synthetic.

The term “sterol-modified amphiphilic lipid” as used herein refers toamphiphilic lipid compounds having a hydrophilic head group, and two ormore hydrophobic tails of which at least one is sterol. “Sterol-modifiedamphiphilic phospholipids” refers to a sterol-modified amphiphilic lipidcomprising a phosphate-containing moiety, such as phosphocholine orphosphoglycerol.

The term “therapeutic agent” as used herein refers to an agent used intesting, development, or application as a therapeutic, including drugsand pharmaceutical agents.

The term “drug” as used herein refers to any chemical compound (e.g., apeptide epoxyketone compound) used in the diagnosis, treatment, orprevention of disease or other abnormal condition.

The term “prodrug” as used herein refers to compounds that, underphysiological conditions, are converted into therapeutically activeagents. A common method for making a prodrug is to include selectedmoieties that are hydrolyzed under physiological conditions to revealthe desired molecule. In other embodiments, the prodrug is converted byan enzymatic activity of the subject.

The terms “therapeutically acceptable” and “pharmaceutically acceptable”as used herein refer to a material that is not biologically or otherwiseundesirable, i.e., the material can be administered to a subjecttogether with an active ingredient without causing undesirablebiological effects or interacting adversely with any other component ofthe composition.

The term “emulsion” as used herein refers to a mixture of two immiscible(unblendable) substances.

The term “bilayer” as used herein refers to a structure composed ofamphiphilic lipid molecules (often phospholipids) arranged in twomolecular layers, with the hydrophobic tails on the interior and thepolar head groups on the exterior surfaces.

The term “monolayer” as used herein refers to a single molecular layerof amphipathic molecules with the head groups aligned on one side, andhydrophobic groups on the opposite side.

The term “liposome” as used herein refers to a vesicle comprising alipid bilayer, for example, a closed vesicle formed when phospholipidsor their derivatives are dispersed in water. The liposomes of thepresent invention typically comprise one or more phospholipids, and mayalso contain mixed lipid chains with surfactant properties (e.g., eggphosphatidylethanolamine). Liposome can employ surface ligands to targetbinding to unhealthy tissue (e.g., tumors or neoplastic cells).Liposomes typically have an aqueous core.

The term “entrapped” as used herein refers to the non-covalentassociation of peptide epoxyketone compounds with a liposome bilayerand/or the liposome's interior aqueous volume (also called theliposome's aqueous core).

The terms “liposomal composition” and “liposome-containing composition”are used interchangeably herein and refer to liposome formulations ormixtures comprising lipids and peptide epoxyketone compounds, and suchliposome mixtures or formulations can further comprise additionalexcipients. A liposomal composition typically comprises an aqueoussolution comprising the liposomes. Encapsulated aqueous solution isaqueous solution in the aqueous core of the liposomes. Non-encapsulatedaqueous solution is aqueous solution in which the liposomes aredispersed.

The term “excipient” as used herein typically refers to anypharmacologically inactive substance used for in the formulation oradministration of the liposomal compositions of the present invention,for example, phospholipid, buffer, a carrier or vehicle (such asdiluents), and so on. Examples of excipients useful in the practice ofthe present invention are described herein.

The term “pH adjusting agent” as used herein refers to any agent used tomodify the pH of an aqueous solution. pH is adjusted by using acidifying(e.g., acids) and alkalizing agents (e.g., salts of acids or bases).Acidifying agents are used in a formulation to lower the pH andalkalizing agents are used to increase the pH. pH adjusting agentsinclude buffering systems (e.g., combinations of acids and bases).Pharmaceutical compositions of the present invention can contain one ormore of these agents to achieve a desirable pH either for preparation(i.e., in bulk solution) of the composition or upon reconstitution fortherapeutic administration.

The term “solublizing agent” as used herein refers to an agent,typically a compound, pH adjusting agent, or cosolvent, that increasesthe solubility of a peptide epoxyketone compound in an aqueous solution.

The term “physiological conditions” as used herein refers to conditionscompatible with living cells, e.g., predominantly aqueous conditions ofa temperature, pH, salinity, etc.

The terms “therapeutic composition,” “pharmaceutical composition,”“therapeutic preparation,” and “pharmaceutical preparation” are usedinterchangeably herein and encompass liposomal compositions of thepresent invention suitable for application or administration to asubject, typically a human. In general such compositions are safe,sterile or asceptic, and preferably free of contaminants that arecapable of eliciting undesirable responses in the subject (i.e., thecompound(s) comprising the composition are pharmaceutically acceptable).Compositions can be formulated for application or administration to asubject in need thereof by a number of different routes ofadministration including oral (i.e., administered by mouth or alimentarycanal) or parenteral (e.g., buccal, rectal, transdermal, transmucosal,subcutaneous, intravenous, intraperitoneal, intradermal, intratracheal,intrathecal, pulmonary, and the like).

The term “aseptic conditions ” as used herein typically refers tomanufacturing or processing conditions wherein the manufactured productis free from contamination with pathogens.

The term “subject” as used herein refers to any member of the subphylumchordata, including, without limitation, humans and other primates,including non-human primates such as rhesus macaque, chimpanzees andother apes and monkey species; farm animals such as cattle, sheep, pigs,goats and horses; domestic mammals such as dogs and cats; laboratoryanimals including rodents such as mice, rats and guinea pigs; birds,including domestic, wild and game birds such as chickens, turkeys andother gallinaceous birds, ducks, geese; and the like. The term does notdenote a particular age. Thus, adult, young, and newborn individuals areintended to be covered.

2.0.0 General Overview of the Invention

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular types ofliposomes, particular sources of drugs, particular lipids, particularpolymers, and the like, as use of such particulars can be selected inview of the teachings of the present specification. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Peptide epoxyketone compounds are proteasome inhibitors useful for thetreatment of a wide variety of diseases and conditions. Many peptideepoxyketone compounds (e.g., carfilzomib) have poor water solubility(e.g., carfilzomib is essentially insoluble in water). At present,carfilzomib for injection is prepared by dissolving carfilzomib drugsubstance in sulfobutylether beta cyclodextrin (SBE-β-CD) with citricacid using a slurry method to create a bulk solution that is thenlyophilized to yield a lyophilized carfilzomib suitable forreconstitution and injection.

However, intravenous administration of the carfilzomib SBE-β-CDcomposition results in a short half-life due to rapid metabolism.Clearance of carfilzomib is largely extrahepatic, and carfilzomib ispredominantly eliminated by peptidase cleavage and epoxide hydrolysis.Therefore, multiple weekly injections are used for treatment regimens.In addition, the use of the SBE-β-CD composition can limit doseincreases of carfilzomib, which can impact its best profile activities.

Liposomes are spherical vesicles, typically comprising phospholipids,that have an internal aqueous volume that is enclosed by one or moreconcentric lipid bilayers with the polar head groups oriented towardsthe interior and exterior aqueous phases. Phospholipids arebiocompatible and biodegradable as they are naturally occurring in thebody and are a major constituent of cell membranes. Liposomes can act asdrug carriers by entrapping drugs in the aqueous core and/or within thelipid bilayers. Liposomes range in size and can exist as unilamellar ormultilamellar vesicles.

The present application describes development of a variety of liposomalcompositions incorporating sparingly soluble hydrophobicepoxyketone-based proteasome inhibitors. In some aspects, entrapment ofpeptide epoxyketone compounds is described. In other aspects,incorporation of peptide epoxyketone compounds into the interior aqueouscore of liposomes is described. The liposomal compositions comprisingpeptide epoxyketone compounds described herein maximize the therapeuticwindow of peptide epoxyketone compounds by improving tolerability,efficacy and in vivo half-life.

The pharmaceutical liposomal compositions of the present invention areeither sterile or asceptic and methods of making the pharmaceuticalliposomal compositions are typically carried out under sterile orasceptic conditions. Terminal sterilization of the pharmaceuticalliposomal compositions of the present invention can also be employed.

In a first aspect, the present invention relates to pharmaceuticalliposomal compositions. In some embodiments, the pharmaceuticalliposomal compositions comprise liposome entrapped peptide epoxyketonecompound, wherein (i) liposomes of the liposomal composition compriseone or more lipids (e.g., L-α-phosphatidylcholine,1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and/or sphingomyelin),and in the liposomes the weight ratio of peptide epoxyketonecompound:lipid is between about 0.01:1 and about 1:1. In a preferredembodiment, the weight ratio of peptide epoxyketone compound:lipid isbetween about 0.05:1 to about 0.5:1. Typically, the liposomes have anaverage size of between about 0.05 microns to about 0.5 microns,preferably an average size of between about 0.05 microns to about 0.2microns. Pharmaceutical liposomal compositions of the present inventiontypically comprise an aqueous solution in which liposomes are dispersed.

Embodiments of the pharmaceutical liposomal compositions presentinvention include, but are not limited to, the following: wherein thelipids of the liposomes comprise between about 20 to about 100 weightpercent phospholipids; wherein the lipids of the liposomes furthercomprise between about 10 and about 50 weight percent cholesterol;wherein the lipids of the liposomes comprise between about 1 and about20 weight percent of a hydrophilic polymer-derivatized lipid; andcombinations thereof.

In some embodiments, the lipid of the hydrophilic polymer-derivatizedlipid is cholesterol or a phospholipid.

In some embodiments, the hydrophilic polymer of a hydrophilicpolymer-derivatized lipid is a polyethylene glycol.

In a preferred embodiment, the hydrophilic polymer-derivatized lipid is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (mPEG2000DSPE).

The lipids of the liposomes, in some embodiments, further compriseα-tocopherol, for example, at about 0.001 to about 5 weight percent.

The aqueous solution in which the liposomes are dispersed can alsocomprise one or more excipients, including, but not limited to, a pHadjusting agent (e.g., a buffer) and/or an agent to maintainisotonicity.

In other embodiments of this first aspect of the present invention, theliposomal composition comprises liposomes comprising the peptideepoxyketone compound and a solubilizing agent (e.g., a compound) in aninternal aqueous core of the liposomes. In some embodiments, thesolubilizing agent is a compound (e.g., a cyclodextrin), and theliposomes of the liposomal composition comprise the peptide epoxyketonecompound complexed with the compounds (e.g., a cyclodextrin) in theinternal aqueous core of the liposomes. A preferred solubilizing agentthat is a compound is a cyclodextrin, for example, asulfobutylether-betacyclodextrin or a hydroxypropyl-betacyclodextrin.

The pharmaceutical liposomal composition of the present invention canalso include liposomal compositions wherein the aqueous solution isadjusted to between about pH 3.5 and about pH 7.0. Preferably, theaqueous solution is adjusted to a human physiological pH.

Examples of peptide epoxyketone compounds for use in liposomalcompositions of the present invention include, but are not limited to,compound I. Preferred peptide epoxyketone compounds for use in liposomalcompositions include compound II, compound III, compound IV, and, mostpreferably carfilzomib (compound V).

Preferred embodiments of pharmaceutical liposomal compositionscomprising peptide epoxyketone compounds include, but are not limitedto, the following: peptide epoxyketonecompound-EPC-mPEG2000DSPE-cholesterol; and peptide epoxyketonecompound-sphingomyelin-mPEG2000DSPE-cholesterol.

In a second aspect the present invention relates to dry pharmaceuticalcompositions formed by drying the pharmaceutical liposomal compositionsdescribed herein. One embodiment of this second aspect of the presentinvention is a dry pharmaceutical composition comprising one or morelipids, the lipids comprising at least one phospholipid selected fromthe group consisting of L-α-phosphatidylcholine,1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and sphingomyelin,wherein the weight ratio of peptide epoxyketone compound:lipid isbetween about 0.01:1 and about 1:1. In some embodiments, drypharmaceutical compositions further comprise additional excipients, forexample cryoprotectant agents (e.g., glycerol, dimethylamine,dimethylsulfoxide), glass transition modifying agents (e.g. sugars,polyols, polymers, amino acids), combinations thereof, and/or otherstabilizing excipients.

In a third aspect, the present invention relates to a method of making apharmaceutical liposomal composition, the method comprising: preparing adried film comprising a peptide epoxyketone compound and at least onelipid (e.g., a phospholipid such as L-α-phosphatidylcholine,1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and/or sphingomyelin),wherein the weight ratio of peptide epoxyketone compound:lipid isbetween about 0.01:1 and about 1:1 (preferably the weight ratio ofpeptide epoxyketone compound:lipid is between about 0.05:1 to about0.5:1); and rehydrating the dried film with an aqueous solution to forma liposomal composition comprising liposomes and an aqueous solution.

In preferred embodiments, the method further comprises sizing theliposomes to have an average size of between about 0.05 microns to about0.5 microns (preferably an average size of between about 0.05 microns toabout 0.2 microns).

Embodiments of the methods of this aspect of the present invention formaking pharmaceutical liposomal compositions include, but are notlimited to, the following: wherein the lipids of the dried film comprisebetween about 20 to about 100 weight percent phospholipids; wherein thelipids of the dried film further comprise between about 10 and about 50weight percent cholesterol; wherein the lipids of the dried film furthercomprise between about 1 and about 20 weight percent of a hydrophilicpolymer-derivatized lipid; and combinations thereof.

In some embodiments, the lipid of the hydrophilic polymer-derivatizedlipid is cholesterol or a phospholipid.

In some embodiments, the hydrophilic polymer of a hydrophilicpolymer-derivatized lipid is a polyethylene glycol.

In a preferred embodiment, the hydrophilic polymer-derivatized lipid is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000].

The lipids of the dried film, in some embodiments, further compriseα-tocopherol, for example, at about 0.001 to about 5 weight percent.

In some embodiments, the method further comprises adding one or moreexcipients, including, but not limited to, a pH adjusting agent (e.g., abuffer) and/or an agent to maintain isotonicity, to the aqueous solutionin which the liposomes are dispersed.

In some embodiments, the method further comprises adjusting the pH ofthe aqueous solution to between about pH 3.5 and about pH 7.0,preferably adjusting the pH of the aqueous solution to a humanphysiological pH.

Examples of peptide epoxyketone compounds for use in the method ofmaking liposomal compositions of the present invention include, but arenot limited to, compound I. Preferred peptide epoxyketone compounds foruse in liposomal compositions include compound II, compound III,compound IV, and, most preferably carfilzomib (compound V).

In a fourth aspect, the present invention relates to pharmaceuticalliposomal compositions made by the methods of the third aspect of theinvention.

In a fifth aspect, the present invention relates to a method of making apharmaceutical liposomal composition comprising preparing a dried filmcomprising one or more lipids and rehydrating the dried film with anaqueous solution comprising a peptide epoxyketone compound. Typicallythe method comprises preparing a dried film comprising at least onephospholipid (e.g., L-α-phosphatidylcholine,1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and/or sphingomyelin;and rehydrating the dried film with an aqueous solution comprising apeptide epoxyketone compound and one or more solubilizing agent to forma liposomal composition comprising liposomes dispersed in the aqueoussolution.

In some embodiments of this fifth aspect of the present invention, oneor more solubilizing agent is, for example, a compound, a pH adjustingagent (e.g., an organic or non-organic acid), a cosolvent (e.g., ethanolor dimethylsulfoxide), or a combination thereof. Cyclodextrin(preferably a sulfobutylether-betacyclodextrin or ahydroxypropyl-betacyclodextrin) is an example of a compound used as asolubilizing agent. In some embodiments, the pH adjusting agent is usedto bring the pH of the aqueous solution to between about pH 0.5 and pH3.0, preferably to a pH of about pH 1.0. In some embodiments, afterformation of the liposomes the pH of the liposomal composition isadjusted to between about between about pH 3.5 and about pH 7.0,preferably to about a human physiological pH. In some embodiments, forexample for pH adjustment and/or removal of a cosolvent, the methodfurther comprises processing the liposomal composition using dialysis,desalting, buffer exchange, and/or gel filtration.

The weight ratio of peptide epoxyketone compound:lipid in the liposomesof the liposomal composition can be, for example, between about 0.01:1and about 1:1.

The method can further comprise sizing the liposomes to have an averagesize of between about 0.05 microns to about 0.5 microns, an average sizeof between about 0.05 microns to about 0.2 microns.

Embodiments of the methods of this aspect of the present invention formaking pharmaceutical liposomal compositions include, but are notlimited to, the following: wherein the lipids of the dried film comprisebetween about 20 to about 100 weight percent phospholipids; wherein thelipids of the dried film further comprise between about 10 and about 50weight percent cholesterol; wherein the lipids of the dried film furthercomprise between about 1 and about 20 weight percent of a hydrophilicpolymer-derivatized lipid; and combinations thereof.

In some embodiments, the lipid of the hydrophilic polymer-derivatizedlipid is cholesterol or a phospholipid.

In some embodiments, the hydrophilic polymer of a hydrophilicpolymer-derivatized lipid is a polyethylene glycol.

In a preferred embodiment, the hydrophilic polymer-derivatized lipid is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000].

The lipids of the dried film, in some embodiments, further compriseα-tocopherol, for example, at about 0.001 to about 5 weight percent.

In some embodiments, the method further comprises, after forming theliposomal composition, removing peptide epoxyketone compound from theaqueous solution in which the liposomes are dispersed (that is,non-encapsulated aqueous solution) using, for example, dialysis,ultracentrifugation, gel filtration, or a combination thereof.

In some embodiments, the method further comprises adding one or moreexcipients, including, but not limited to, a pH adjusting agent (e.g., abuffer) and/or an agent to maintain isotonicity, to the aqueous solutionin which the liposomes are dispersed.

In some embodiments, the method further comprises adjusting the pH ofthe aqueous solution to between about pH 3.5 and about pH 7.0,preferably adjusting the pH of the aqueous solution to a humanphysiological pH.

Examples of peptide epoxyketone compounds for use in the method ofmaking liposomal compositions of the present invention include, but arenot limited to, compound I. Preferred peptide epoxyketone compounds foruse in liposomal compositions include compound II, compound III,compound IV, and, most preferably carfilzomib (compound V).

In a sixth aspect, the present invention relates to pharmaceuticalliposomal compositions made by the methods of the fifth aspect of theinvention.

In a seventh aspect, the present invention relates to a method of makinga pharmaceutical liposomal composition comprising preparing a lipidsolution comprising one or more lipids and a solvent and injecting thelipid solution into an aqueous solution comprising a peptide epoxyketonecompound. Typically the method comprises preparing a lipid solutioncomprising a solvent and at least one phospholipid (e.g.,L-α-phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and/or sphingomyelin);and injecting the lipid solution into an aqueous solution comprising apeptide epoxyketone compound and one or more solubilizing agent to forma liposomal composition comprising liposomes dispersed in the aqueoussolution.

In some embodiments the solvent is an organic solvent, for example analcohol (e.g., ethanol).

In some embodiments of this seventh aspect of the present invention, theone or more solubilizing agent is, for example, a compound, a pHadjusting agent (e.g., an organic or non-organic acid), a cosolvent(e.g., ethanol or dimethylsulfoxide), or a combination thereof.Cyclodextrin (preferably a sulfobutylether-betacyclodextrin or ahydroxypropyl-betacyclodextrin) is an example of a compound used as asolubilizing agent. In some embodiments, the pH adjusting agent is usedto bring the pH of the aqueous solution to between about pH 0.5 and pH3.0, preferably to a pH of about pH 1.0.

In some embodiments, for example for pH adjustment and/or removal of asolvent and/or a cosolvent, the method further comprises processing theliposomal composition using dialysis, desalting, buffer exchange, and/orgel filtration.

The weight ratio of peptide epoxyketone compound:lipid in the liposomesof the liposomal composition can be, for example, between about 0.01:1and about 1:1.

The method can further comprise sizing the liposomes to have an averagesize of between about 0.05 microns to about 0.5 microns, an average sizeof between about 0.05 microns to about 0.2 microns.

Embodiments of the methods of this aspect of the present invention formaking pharmaceutical liposomal compositions include, but are notlimited to, the following: wherein the lipids of the liposomes of theliposomal composition comprise between about 20 to about 100 weightpercent phospholipids; wherein the lipids of the liposomes of theliposomal composition further comprise between about 10 and about 50weight percent cholesterol; wherein the lipids of the liposomes of theliposomal composition further comprise between about 1 and about 20weight percent of a hydrophilic polymer-derivatized lipid; andcombinations thereof.

In some embodiments, the lipid of the hydrophilic polymer-derivatizedlipid is cholesterol or a phospholipid.

In some embodiments, the hydrophilic polymer of a hydrophilicpolymer-derivatized lipid is a polyethylene glycol.

In a preferred embodiment, the hydrophilic polymer-derivatized lipid is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000].

The lipids of the liposomes of the liposomal composition, in someembodiments, further comprise α-tocopherol, for example, at about 0.001to about 5 weight percent.

In some embodiments, the method further comprises, after forming theliposomal composition, removing peptide epoxyketone compound from theaqueous solution in which the liposomes are dispersed (that is,non-encapsulated aqueous solution) using, for example, dialysis,ultracentrifugation, gel filtration, or a combination thereof.

In some embodiments, the method further comprises adding one or moreexcipients, including, but not limited to, a pH adjusting agent (e.g., abuffer) and/or an agent to maintain isotonicity, to the aqueous solutionin which the liposomes are dispersed.

In some embodiments, the method further comprises adjusting the pH ofthe aqueous solution to between about pH 3.5 and about pH 7.0,preferably adjusting the pH of the aqueous solution to a humanphysiological pH.

Examples of peptide epoxyketone compounds for use in the method ofmaking liposomal compositions of the present invention include, but arenot limited to, compound I. Preferred peptide epoxyketone compounds foruse in liposomal compositions include compound II, compound III,compound IV, and, most preferably carfilzomib (compound V).

In an eighth aspect, the present invention relates to pharmaceuticalliposomal compositions made by the methods of the seventh aspect of theinvention.

In a ninth aspect, the present invention relates to methods of treatinga disease or condition in a subject in need of treatment, comprisingadministering a therapeutically effective amount of a pharmaceuticalliposomal composition, as described herein, comprising liposomescomprising a peptide epoxyketone compound. In some embodiments themethods of treating further comprise simultaneous, sequential, orseparate administration of a therapeutically effective amount of anothertherapeutic agent, for example, a chemotherapeutic agent, a cytokine, asteroid, an immunotherapeutic agent, or combinations thereof. Examplesof diseases or conditions that are treated using the liposomalcompositions of the present invention comprising peptide epoxyketonecompounds include, but are not limited to, multiple myeloma, solidtumors, infections, and autoimmune diseases.

In another aspect, the present invention includes dry pharmaceuticalcompositions comprising one or more lipids and a peptide epoxyketonecompound. Such dry pharmaceutical compositions can be rehydrated for usein the methods of the present invention. In one embodiment, a drypharmaceutical composition comprises, one or more amphipathic lipids(e.g., lipids comprising at least one phospholipid), and (ii) a peptideepoxyketone compound. Dry pharmaceutical compositions can be made usingany of the liposomal compositions of the present invention describedherein (e.g., compositions further comprising a solubilizing agent), aswell as liposomal compositions comprising peptide epoxyketone compoundsmade by any of the methods described herein. In some embodiments, drypharmaceutical compositions further comprise additional excipients, forexample cryoprotectant agents (e.g., glycerol, dimethylamine,dimethylsulfoxide), glass transition modifying agents (e.g. sugars,polyols, polymers, amino acids), and/or other stabilizing excipients.

3.0.0 Pharmaceutical Compositions

The present invention relates to liposomal compositions of peptideepoxyketones compounds (e.g., carfilzomib) and prodrugs thereof, andmethods of making and using such compositions.

3.1.0 Peptide Epoxyketone Compounds

Examples of peptide epoxyketone compounds useful in the practice of thepresent invention are described in U.S. Pat. No. 7,417,042, and include,but are not limited to, a peptide epoxyketone compound having thestructure of formula I:

wherein X is O, NH, or N-alkyl; Y is NH, N-alkyl, O, or C(R⁹)₂; Z is Oor C(R⁹)₂; R¹, R², R³, and R⁴ are all hydrogen; each R⁵, R⁶, R⁷, R⁸, andR⁹ is independently selected from hydrogen, C₁₋₆alkyl, C₁₋₆hydroxyalkyl,C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, each of which is optionallysubstituted with a group selected from alkyl, amide, amine, carboxylicacid or a pharmaceutically acceptable salt thereof, carboxyl ester,thiol, and thioether; m is an integer from 0 to 2; and n is an integerfrom 0 to 2. Terms used to describe these compounds are further setforth in the “Definitions” section.

Examples of specific peptide epoxyketone compounds useful in thepractice of the present invention include the following compounds havingformulas II, III, and IV (“Ph” in the following compounds represents aphenyl group):

In a preferred embodiment of the present invention, the peptideepoxyketone compound is carfilzomib having formula V:

In the liposomal compositions of the present invention, the weight ratioof peptide epoxyketone compound:total lipid in the liposomes is betweenabout 0.01:1 and about 1:1, preferably between about 0.025:1 to about0.5:1, and more preferably between about 0.05:1 to about 0.25:1.

3.2.0 Liposome Components

Types of lipids used in the practice of the present invention include,but are not limited to phospholipids, sterols, and modifications andderivatives thereof. Additional amphipathic lipids can also be used inthe practice of the present invention.

Preferred vesicle forming amphipathic lipids for use in the practice ofthe present invention include phospholipids and derivatives thereof.Phospholipids fall generally into three classes, neutral, cationic, andanionic. Examples of phospholipids useful in the practice of the presentinvention include, but are not limited to, the following:phosphatidylcholine; L-α-phosphatidylcholine (egg phosphatidylcholine(EPC), or hydrogenated soy phosphatidylcholine (HSPC));1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); phosphatidylserine(PS); phosphatidylinositol (PI); phosphatidylglycerol (PG);phosphatidylethanolamine (PE); dioleoyl phosphatidylglycerol (DOPG);dioleoyl phosphatidylcholine (DOPC); dioleoyl phosphatidylserine (DOPS);1,2-dileoyl-sn-3-phosphoethanolamine (DOPE); diacylphosphatidylcholine;diacylphosphatidic acid; N-dodecanoyl phosphatidylethanolamine:N-succinyl phosphatidylethanolamine: N-glutarylphosphatidylethanolamine: lysylphosphatidylglycerol; sphingolipids(e.g., sphingomyelin); and mixtures thereof.

Further lipids useful in the practice of the present invention include,for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC);N-(2,3-dioleyloxy)propyl-N,N-N-triethylammonium chloride (DOTMA);N,N-distearyl-N,N-dimethylammonium bromide (DDAB);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP);N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (DOSPA); dioctadecylamidoglycylcarboxyspermine (DOGS);N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE); stearylamine; dicetyl phosphate; β-oleoyl-γ-palmitoyl;and mixtures thereof.

Preferred lipids for use in the practice of the present inventioninclude, but are not limited to: L-α-phosphatidylcholine (e.g., eggphosphatidylcholine (EPC), or hydrogenated soy phosphatidylcholine(HSPC)); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). In some embodiments,the lipids of the liposomes comprise between about 20 to about 100weight percent phospholipid, preferably between about 30 and about 90weight percent phospholipid.

A variety of sterols and derivatives thereof (e.g., cholesterol) can beused to stabilize liposomes. Sterol-modified amphiphilic lipids areknown in the art (see, e.g., U.S. Patent Application Publication No.2011/0177156). Sterols for use in the practice of the present invention,such as cholesterol, also can be derivatized with a variety ofhydrophilic polymers (PEG-cholesterol derivatives; see, e.g., U.S. Pat.No. 6,270,806). In an embodiment of the present invention, sterols orderivatives thereof can be added to the liposomal composition tostabilize the lipid bilayer. Preferred sterols for use in the practiceof the present invention are cholesterol and its derivatives (e.g.,cholesterol hemisuccinate), for example, the lipids of the liposomes ofthe liposomal compositions of the present invention can comprise betweenabout 10 and about 50 weight percent cholesterol, preferably betweenabout 15 and about 40 weight percent cholesterol.

In other embodiments, cholesterol is chemically modified with a liganddesigned to be recognized by a particular organ or cell type such as along chain fatty acid, an amino acid, an oligosaccharide, a hormone, anamino acid derivative, a protein, glycoprotein, modified protein, or thelike. The resultant liposome is suitable for being targeted to aspecific organ or cell type (see, e.g., U.S. Pat. No. 4,544,545).

Additional examples of liposomal compositions including targetingfactors that can be used, in view of the teachings of the presentspecification, include U.S. Pat. Nos. 5,049,390; 5,780,052; 5,786,214;5,830,686; 6,056,973; 6,110,666; 6,177,059; 6,245,427; 6,316,024;6,524,613; 6,530,944; 6,749,863; 6,803,360; 6,960,560; 7,060,291;7,101,985; and U.S. Patent Application Nos. 2002/0198164; 2003/0027779;2003/0220284; 2003/0224037; 2003/0228285; 2003/143742; and 2004/0022842.

Steric stabilization refers to the colloidal stability conferred on theliposome by a variety of hydrophilic polymers or hydrophilicglycolipids, for example, polyethylene glycol and the ganglioside GM1.Liposomes can contain PEG-PE, GM1, or another such glycolipid or polymerthat demonstrates a relatively long half-life in the generalcirculation. Hydrophilic polymers such as PEG and other polyethoxylatedpolymers can be used to shield liposomes to enhance the circulatoryhalf-life of the liposome. Such hydrophilic polymers can be associatednon-covalently with the liposomes or conjugated or covalently linked toa particular component of the liposome (e.g., PEG-modified lipids;1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt) (mPEG-DSPE)). Additional exemplaryhydrophilic polymers include, but are not limited to, polyvinyl alcohol,polylactic acid, polyglycolic acid, polyvinylpyrrolidone,polyacrylamide, polyglycerol, polyaxozlines, and mixtures thereof.

In some embodiments of the liposomal compositions described herein, thelipids of the liposomes can comprise between about 0.1 and about 30weight percent, preferably between about 1 and about 20 weight percentof a hydrophilic polymer-derivatized lipid. Preferred hydrophilicpolymers for use in the practice of the present invention arepolyethylene glycols (e.g., phospholipids conjugated to monomethoxypolyethylene glycol, for example,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N4methoxy(polyethyleneglycol)-2000] (mPEG-DSPE)).

Additional examples of liposomal compositions that can be used, in viewof the teachings of the present specification, include: U.S. Pat. Nos.4,789,633; 4,925,661; 4,983,397; 5,013,556; 5,534,241; 5,593,622;5,648,478; 5,676,971; 5,756,069; 5,834,012; 5,846,458; 5,891,468;5,945,122; 6,056,973; 6,057,299; 6,077,834; 6,126,966; 6,153,596;6,287,593; 6,316,024; 6,387,397; 6,476,068; 6,586,559; 6,627,218;6,723,338; 6,897,196; 6,936,272; 6,960,560; 7,122,202; 7,311,924;7,361,640; and 7,901,708; and U.S. Patent Application Publication Nos.2003/0072794; 2003/0082228; 2003/0166601; 2003/0203865 2003/0215490;2003/0224037; 2004/0022842; 2004/0234588; and 2005/0136064.

The liposomal compositions typically comprise liposome entrapped peptideepoxyketone compounds and an aqueous carrier.

Typical excipients useful in the practice of the present inventioninclude, but are not limited to, the following: carrier or vehicle(e.g., water or buffered aqueous solutions); pH adjusting agents;antioxidants (e.g., α-tocopherol, methionine, ascorbic acid, sodiumthiosulfate, ethylenediaminetetraacetic acid, citric acid, cysteins,thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxanisol,butylated hydroxyltoluene, and propyl gallate, and mixtures thereof);agents to maintain isotonicity (e.g., sodium chloride, sugars, polyols(sugar alcohols), boric acid, sodium tartrate, propylene glycol, andmixtures thereof); one or more sugars (e.g., trehalose, maltose,sucrose, lactose, mannose, dextrose, fructose, etc.) or sugar alcohol(e.g., sorbitol, maltitol, lactitol, mannitol, glycerol, etc.), alcohol(e.g., ethanol, t-butanol, etc.); and preservatives (alcohols, benzoicacid, salicylic acid, phenol and its derivatives (e.g., cresol,p-cresol, m-cresol and o-cresol), cetrimide, BHA (butylatedhydroxytoluene), BHA (butylated hydroxyanisole); and mixtures thereof).

pH adjusting agents useful in the practice of the present inventioninclude, but are not limited to hydrochloric acid, sodium hydroxide,citric acid, phthalic acid, acetic acid, ascorbic acid, phosphate,glutamate, sodium or potassium succinate, tartrate, histidine, sodium orpotassium phosphate, Tris(tris(hydroxymethyl)aminomethane), anddiethanolamine. Buffers comprising both acids and bases/salts can alsobe used.

In a preferred embodiment of the present invention, the liposomes can berehydrated using buffered aqueous solutions (e.g., phosphate buffersaline (PBS)), 0.9% Saline, 5% Dextrose, 10% Sucrose, or water forinjection (WFI) as the rehydration medium. In some embodiments, the pHof the aqueous phase of the liposomal compositions is adjusted, forexample, to approximately human physiological pH (i.e., between about pH6.5 and about pH 7.5). Excipients typically present in the aqueous phaseinclude, but are not limited to, buffer systems, agents to maintainisotonicity, sugars, sugar alcohols, and/or preservatives.

Exemplary embodiments of liposomal compositions of peptide epoxyketonesinclude, but are not limited to, the following: carfilzomib-EPC;carfilzomib-EPC-cholesterol; carfilzomib-DSPC;carfilzomib-DSPC-cholesterol; carfilzomib-DPPC;carfilzomib-DPPC-cholesterol; carfilzomib-sphingomyelin;carfilzomib-sphingomyelin-cholesterol.

Further examples comprise PEGylated liposomal compositions of peptideepoxyketones that include, but are not limited to, the following:carfilzomib-EPC-mPEG2000DSPE; carfilzomib-EPC-mPEG2000DSPE-cholesterol;carfilzomib-DSPC-mPEG2000DSPE;carfilzomib-DSPC-mPEG2000DSPE-cholesterol;carfilzomib-sphingomyelin-mPEG2000DSPE; andcarfilzomib-sphingomyelin-mPEG2000DSPE-cholesterol.

Preferred embodiments of liposomal compositions of peptide epoxyketonesinclude, but are not limited to, the following:carfilzomib-EPC-mPEG2000DSPE-cholesterol; andcarfilzomib-sphingomyelin-mPEG2000DSPE-cholesterol.

Examples of specific embodiments of liposomal compositions are set forthin Examples 1, 7, 10, and 11.

4.0.0 Preparing Liposomal Compositions

Liposomes can be prepared by a variety of techniques (e.g., Szoka, F.,Jr., et al., “Comparative Properties and Methods of Preparation of LipidVesicles (Liposomes),” Annual Review of Biophysics and Bioengineering,June 1980, 9:467-508; U.S. Pat. No. 4,235,871) including reverse phaseevaporation methods. The reverse phase evaporation vesicles initiallyhave typical average sizes between about 2-4 microns.

In some embodiments, liposomes are formed by simple lipid-film hydrationtechniques (see, e.g., Examples 1 and 2). In this procedure, a mixtureof liposome-forming lipids of the type described herein and peptideexpoxyketone compounds are dissolved in a suitable organic solvent andevaporated in a vessel to form a thin film, which is then covered by anaqueous medium. The lipid film hydrates to form vesicles typically withsizes between about 0.1 to 10 microns.

Other embodiments of the present invention include, a method ofpassively encapsulating a hydrophobic, water-insoluble, peptideexpoxyketone compound into the internal aqueous core of the liposome.Such encapsulation in the aqueous core can be facilitated using one ormore solubilizing agent. Solubilizing agents increase the solubility ofa peptide expoxyketone compound in an aqueous solution. Solubilizingagents include, for example, compounds to facilitate solubilization(e.g., cyclodextrin), pH adjusting agents, cosolvents, and combinationsthereof. Advantages of encapsulating peptide expoxyketone compounds inthe interior aqueous core of liposomes include greater protection fromchemical and biological degradation, slower diffusion, greater tissuedistribution and extended drug release profiles.

Cyclodextrins are an example of compounds to facilitate solubilizationof peptide expoxyketone compounds in aqueous solution. Cyclodextrins canbe charged or neutral, native (cyclodextrins α, β, γ, δ, ε), branched orpolymerized. In certain aspects, cyclodextrins can be chemicallymodified, for example, by substitution of one or more hydroxypropyls bygroups such as alkyls, aryls, arylalkyls, glycosidics, or byetherification, esterification with alcohols or aliphatic acids. Fromthese groups, particular preference is given to those fromhydroxypropyl, methyl m, sulfobutylether groups (see, e.g., Stella V.J., et al., Toxicol. Pathol. 36(1):30-42 (2008)). In certain aspects,cyclodextrins comprise six, seven, or eight glucopyranose units.

Cyclodextrins include α-cyclodextrin,β-cyclodextrin, and γ-cylcodextrin.Suitable α-cyclodextrins include but are not limited tohydroxypropyl-α-cyclodextrin and hydroxyethyl-α-cyclodextrin. Suitableβ-cyclodextrins include but are not limited tohydroxypropyl-β-cyclodextrin (e.g., such as 2-hydroxypropylcyclodextrin), carboxymethyl-β-cyclodextrin,dihydroxypropyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin,2,6-di-O-methyl-β-cyclodextrin, methyl-β-cyclodextrin, randomlymethylated cylcodextrin, and sulfated-β-cyclodextrin. Suitableγ-cyclodextrins include hydroxypropyl γ-cyclodextrin,dihydroxypropyl-γ-cyclodextrin, hydroxyethyl γ-cyclodextrin, andsulfated-γ-cyclodextrin.

Preferred cyclodextrins for use in the practice of the present inventioninclude β-cyclodextrins (such as sulfobutyl ether-β-cyclodextrins(abbreviated as SBE-β-CD or SBE-CD; CAPTISOL® (Ligand Pharmaceuticals,Inc., La Jolla, Calif.)); or hydroxypropyl-betacyclodextrin (HP-β-CD;Janssen, Titusville N.J.; see also Gould S, et al., Food Chem.Toxicol.43(10):1451-9 (2005)); see also U.S. Pat. Nos. 4,920,214;5,385,891; 5,718,905; and 6,046,177).

Peptide expoxyketone compounds are typically hydrophobic and have lowsolubility in water. Peptide expoxyketone compounds have increasedaqueous solubility in acidic solutions. Accordingly, lowering the pH ofthe aqueous solution in which a peptide expoxyketone compound is beingdissolved can enhance aqueous solubilization. For example, the pH of theaqueous solution can be lowered using a pH adjusting agent to a pH ofless than ˜pH 2 using an acid, for example, hydrochloric acid. Examplesof pH adjusting agents are listed above. Preferred pH adjusting agentsfor solubilization of peptide epoxyketone compounds include, but are notlimited to, hydrochloric acid, citric acid, methanesulfonic acid,sulfuric acid, tartaric acid, acetic acid, and/or maleic acid. Apreferred pH for solubilization is typically ˜pH 1.

Further, solubility of peptide expoxyketone compounds in aqueoussolutions can be increased by the use of cosolvent solubilization.Examples of cosolvents as solubilizing agents include, but are notlimited to, dimethylsulfoxide, methylpyrrolidone,dimethylimidazolidinone, tetrahydrofuran, N,N-dimethylacetamide,propylene glycol, benzyl alcohol, polyethylene glycol, ethanol, andcombinations thereof.

As noted above, solubility of peptide expoxyketone compounds in aqueoussolutions can be increased by use of solubilizing agents, including, butnot limited to, compounds, pH adjusting agents, cosolvents, andcombinations thereof.

In some embodiments, liposomes are formed by a thin film hydrationmethod followed by rehydration using an aqueous solution comprising apeptide expoxyketone compound and solubilizing agent. In such a method,a lipid film is formed wherein the lipid film comprises, for example,any one or combination of phospholipids, including but not limited tothe following: L-α-phosphatidylcholine (egg phosphatidylcholine, EPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), andsphingomyelin (SPH); phospholipids conjugated to monomethoxypolyethylene glycol (PEG); and cholesterol. The lipids are typicallydissolved in an organic solvent (e.g., Methanol:Chloroform) followed bysolvent removal to form a lipid film.

The peptide expoxyketone compound is solubilized in an aqueous solutioncomprising, for example, about 1% to about 60% (w/w), preferably about5% to about 40% of a solubilizing agent (e.g.,sulfobutylether-betacyclodextrin or hydroxypropyl-betacyclodextrin). Theaqueous solution can also include, for example, a pH adjusting agent(e.g., citrate buffer, ˜pH 3, or Glycine-HCl; ˜pH 2) and/or a cosolventfor solubilization of the peptide expoxyketone compound. The aqueousdrug solution is used to rehydrate the lipid film. Upon rehydration,self-assembling vesicles form concentric lipid bilayers encapsulating aninternal aqueous volume (i.e., aqueous core) of the aqueous solutioncomprising the peptide expoxyketone compound. The unencapsulated freedrug can be removed, for example, by centrifugation and the liposomalcomposition washed, for example, with phosphate buffer saline. Example 7describes making liposomal compositions following this method.

In other embodiments, liposomes are formed by a lipid solution injectionmethod wherein a lipid solution is injected into an aqueous solutioncomprising a peptide expoxyketone compound. This method typicallycomprises solubilizing a peptide expoxyketone compound (e.g., indifferent solid states, such as crystalline or amorphous), using pHcontrol, with or without, cosolvent solubilization in an aqueoussolution. The lipids are dissolved in a solvent, for example, ethanol,followed by injection into the aqueous solution comprising the peptideexpoxyketone compound while stirring. Liposome vesicles are formed uponinjection into the aqueous solution trapping small amounts of aqueoussolution in the internal aqueous compartment(s) of the vesicles. Example10 describes making liposomal compositions following this method.

In some embodiments, for example for pH adjustment and/or removal ofsolvent and/or a cosolvent, the methods of the invention furthercomprise processing the liposomal composition using dialysis, desalting,buffer exchange, and/or gel filtration.

A liposomal composition of the present invention generally contains anon-homogenous mixture of lipids, peptide epoxyketone compound, andaqueous solution, wherein the liposomes are of substantially homogenoussize, with an average size of less than about 1 micron, preferablybetween about 0.01 to about 1.0 microns, and more preferably betweenabout 0.05 and about 0.5 microns. In some embodiments, liposomes of theliposomal compositions of the present invention have average diametersof less than about 0.2 microns. Sizing serves to eliminate largerliposomes and to produce a defined size range having optimalpharmacokinetic properties.

One effective sizing method for vesicles involves extruding an aqueoussuspension of the liposomes through a series of polycarbonate membraneshaving a selected uniform pore size in the range of 0.03 to 0.2 micron,typically 0.05, 0.08, 0.1, or 0.2 microns. The pore size of the membranecorresponds roughly to the largest sizes of liposomes produced byextrusion through that membrane, particularly where the preparation isextruded two or more times through the same membrane. The liposomes canbe extruded through successively smaller-pore membranes, to achieve agradual reduction in liposome size. This method of liposome sizing isused in preparing homogeneous-size vesicle compositions. A more recentmethod involves extrusion through an asymmetric ceramic filter (see,e.g., U.S. Pat. No. 4,737,323). Homogenization methods are also usefulfor down-sizing liposomes to sizes of 0.1 micron or less.

Sonicating a liposome suspension either by bath or probe sonication canbe used to produce progressive size reduction down to small unilamellarvesicles (SUVs) less than about 0.05 microns in size. Homogenization isanother method that relies on shearing energy to fragment largeliposomes into smaller ones. In a typical homogenization procedure,vesicles are recirculated through a standard emulsion homogenizer untilselected liposome sizes, typically between about 0.1 and 0.5 microns,are observed. In both methods, the particle size distribution can bemonitored by conventional laser-beam particle size discrimination. Afurther sizing method includes use of a microfluidizer.

Centrifugation and molecular sieve chromatography are other methodsavailable for producing a liposome suspension with particle sizes belowa selected threshold less than 1 micron. These two methods both involvepreferential removal of larger liposomes, rather than conversion oflarge particles to smaller ones.

Examples of preparation, rehydration, and characterization of liposomalcompositions of the present invention are presented in Example 1,Example 2, Example 3, Example 7, Example 10, and Example 11 herein.

In one aspect, the present invention includes methods for thepreparation of the liposomal compositions described herein. In oneembodiment, a method of making a liposomal composition comprises mixing(typically dissolving) lipid and peptide epoxyketone compound in asuitable solvent, evaporating the solvent to produce a dried film,rehydrating the dried film (which in this embodiment comprises lipid andpeptide epoxyketone compound) to form liposomes, and sizing theliposomes. In another embodiment, a method of making a liposomalcomposition comprises a thin film hydration method which produces adried film comprising liposomal components followed by rehydration usingan aqueous solution comprising a peptide expoxyketone compound as wellas a solubilizing agent, a pH adjusting agent, and/or a cosolvent. Inyet another embodiment, a method of making a liposomal compositioncomprises dissolving lipid(s) in solvent(s) and injecting the resultinglipid solution into an aqueous solution comprising a peptideexpoxyketone compound as well as a solubilizing agent, a pH adjustingagent, and/or a cosolvent.

The present invention also includes liposomal compositions comprisingpeptide expoxyketone compounds made by the methods described herein.

Dry pharmaceutical compositions comprising one or more lipids and apeptide epoxyketone compound can be formed from the liposomalcompositions described herein, for example, by lyophilization,desiccation, freeze-drying, spray-drying, or similar method. In someembodiments, dry pharmaceutical compositions further comprise additionalexcipients, for example cryoprotectant agents (e.g., glycerol,dimethylamine, dimethylsulfoxide), glass transition modifying agents(e.g. sugars, polyols, polymers, amino acids), and/or other stabilizingexcipients. Such dry pharmaceutical compositions can be rehydrated foruse in the methods of the present invention. The rehydration media usedfor reconstitution of such dry pharmaceutical compositions can includeexcipients including, but not limited to, a pH adjusting agent, anantioxidant, an agent to maintain isotonicity, a sugar, a sugar alcohol,an alcohol, and/or a preservative.

5.0.0 Uses of the Liposomal Compositions of the Present Invention

In one aspect of the present invention, the liposomal compositionscomprising peptide epoxyketone compounds are useful for the treatment ofcancer. Compounds of the invention also can be used to inhibit NF-κBactivation, and stabilize p53 levels in cell culture.

In one embodiment of the present invention, the liposomal compositionscan be used for anti-inflammatory therapeutic intervention in treatingconditions associated with chronic inflammation, including, but notlimited to COPD, psoriasis, bronchitis, emphysema, and cystic fibrosis.

In another embodiment of the present invention, the liposomalcompositions can be used to treat neurodegenerative diseases andconditions, including, but not limited to: stroke; ischemic damage tothe nervous system; neural trauma (e.g., percussive brain damage, spinalcord injury, and traumatic damage to the nervous system); multiplesclerosis and other immune-mediated neuropathies (e.g., Guillain-Barresyndrome and its variants, acute motor axonal neuropathy, acuteinflammatory demyelinating polyneuropathy, and Fisher Syndrome);HIV/AIDS dementia complex; axonomy; diabetic neuropathy; Parkinson'sdisease; Huntington's disease; multiple sclerosis; bacterial, parasitic,fungal, and viral meningitis; encephalitis; vascular dementia;multi-infarct dementia; Lewy body dementia; frontal lobe dementia suchas Pick's disease; subcortical dementias (such as Huntington orprogressive supranuclear palsy); focal cortical atrophy syndromes (suchas primary aphasia); metabolic-toxic dementias (such as chronichypothyroidism or B12 deficiency); and dementias caused by infections(such as syphilis or chronic meningitis).

In yet another embodiment of the present invention, the liposomalcompositions can be used as a treatment for Alzheimer's disease,comprising administering to a subject an effective amount of peptideepoxyketone-containing liposomal compositions disclosed herein. In suchcases, the liposomal compositions reduce the rate of β-AP processing,reduce the rate of β-AP plaque formation, reduce the rate of β-APgeneration, and reduce the clinical signs of Alzheimer's disease.

Other embodiments of the present invention relate to methods fortreating cachexia and muscle-wasting diseases, cancer, chronicinfectious diseases, fever, muscle disuse (atrophy) and denervation,nerve injury, fasting, renal failure associated with acidosis, diabetes,and hepatic failure. Embodiments of the invention encompass methods for:reducing the rate of muscle protein degradation in a cell; reducing therate of intracellular protein degradation; reducing the rate ofdegradation of p53 protein in a cell; and inhibiting the growth ofp53-related cancers.

Certain embodiments of the present invention relate to a method fortreating hyperproliferative conditions such as diabetic retinopathy,macular degeneration, diabetic nephropathy, glomerulosclerosis, IgAnephropathy, cirrhosis, biliary atresia, congestive heart failure,scleroderma, radiation-induced fibrosis, and lung fibrosis (idiopathicpulmonary fibrosis, collagen vascular disease, sarcoidosis, interstitiallung diseases, and extrinsic lung disorders). The treatment of burnvictims often is hampered by fibrosis; thus, an additional embodiment ofthe invention is the topical or systemic administration of the peptideepoxyketone-containing liposomal composition for burn treatment. Woundclosure following surgery often is associated with disfiguring scars,which can be prevented by inhibition of fibrosis. Thus, in certainembodiments, the invention relates to a method for prevention orreduction of scarring.

Certain embodiments of the present invention relate to a method oftreating ischemia and reperfusion injury, which are associated withhypoxia, a deficiency of oxygen reaching the tissues of the body.Examples of such injuries or conditions include, but are not limited to,acute coronary syndrome (vulnerable plaques), arterial occlusive disease(cardiac, cerebral, peripheral arterial and vascular occlusions),atherosclerosis (coronary sclerosis, coronary artery disease),infarctions, heart failure, pancreatitis, myocardial hypertrophy,stenosis, and restenosis.

Two further embodiments of the present invention are a method forinhibiting or reducing HIV infection in a subject, and a method fordecreasing the level of viral gene expression.

In certain embodiments, compounds of the present invention can be usedfor the inhibition of TNFα to prevent and/or treat septic shock.

An additional embodiment of the present invention is a method forinhibiting antigen presentation in a cell. In such method, the liposomalcomposition is used to treat immune-related conditions such as allergy,asthma, organ/tissue rejection (graft-versus-host disease), andauto-immune diseases, including, but not limited to, lupus, rheumatoidarthritis, psoriasis, multiple sclerosis, and inflammatory boweldiseases (such as ulcerative colitis and Crohn's disease). Thus, afurther embodiment is a method for suppressing the immune system of asubject (e g., inhibiting transplant rejection, allergies, auto-immunediseases, and asthma), including administering to the subject aneffective amount of a compound described herein.

One embodiment of the invention is a method for inhibiting IκB-αdegradation, including contacting the cell with the liposomalcomposition. A further embodiment is a method for reducing the cellularcontent of NF-κB in a cell, muscle, organ, or subject, includingcontacting the cell, muscle, organ, or subject with the liposomalcomposition.

A further embodiment of the invention is a method for treating aproliferative disease in a subject (e.g., cancer, psoriasis, orrestenosis), including administering to the subject an effective amountof the liposomal composition. The invention also encompasses a methodfor treating cyclin-related inflammation in a subject.

Another embodiment of the present invention is a method for treatingp53-related apoptosis.

In a certain embodiments, the invention's liposomal compositions areuseful for the treatment of a parasitic infection, such as infections inhumans caused by a protozoan parasite selected from Plasmodium sps.(including P. falciparum, P. vivax, P. malariae, and P. ovale, whichcause malaria), Trypanosoma sps. (including T. cruzi, which causesChagas' disease, and T. brucei which causes African sleeping sickness),Leishmania sps. (including L. amazonensis, L. donovani, L. infantum, L.mexicana, etc.), Pneumocystis carinii (a protozoan known to causepneumonia in AIDS and other immunosuppressed patients), Toxoplasmagondii, Entamoeba histolytica, Entamoeba invadens, and Giardia lamblia.In certain embodiments, the disclosed compounds are useful for thetreatment of parasitic infections in animals and livestock caused by aprotozoan parasite selected from Plasmodium hermani, Cryptosporidiumsps., Echinococcus granulosus, Eimeria tenella, Sarcocystis neurona, andNeurospora crassa.

In one embodiment of the present invention, the liposomal compositionscan be useful in the treatment and/or prevention of diseases associatedwith bone loss, such as osteoporosis.

Actual dosage levels of peptide epoxyketone compounds in pharmaceuticalcompositions of this invention can be varied so as to obtain an amountof the peptide epoxyketone compound that is effective to achieve thedesired therapeutic response for a particular subject, composition, andmode of administration, without being toxic to the subject.

The concentration of peptide epoxyketone compound in a pharmaceuticallyacceptable mixture will vary depending on several factors, includingdosage of the compound to be administered, pharmacokineticcharacteristics of the compound(s) employed, and route ofadministration. In general, the liposomal compositions of this inventioncan be provided in an aqueous solution for parenteral administration.Typical dose ranges are from about 0.01 to about 50 mg/kg of body weightper day of peptide epoxyketone compound, and can be administered insingle or divided doses. Each divided dose may contain the same ordifferent compounds of the invention. The dosage will be an effectiveamount depending on several factors including the overall health of apatient, and the composition and route of administration of the selectedpeptide epoxyketone compound(s).

Another aspect of the present invention provides a combination treatmentwherein one or more other therapeutic agents are administered with thepeptide epoxyketone-containing liposomal composition. Such combinationtreatment can be achieved by simultaneous, sequential, or separatedosing of the individual components of the treatment.

In certain embodiments of the present invention, a peptideepoxyketone-containing liposomal composition described herein is used aspart of a combination treatment that includes one or more otherproteasome inhibitor(s).

In other embodiments, a liposomal composition of the invention is partof a combination treatment that includes a chemotherapeutic. Suitablechemotherapeutics may include natural products such as vinca alkaloids(i.e., vinblastine, vincristine, and vinorelbine), paclitaxel,epidipodophyllotoxins (i.e., etoposide, teniposide), antibiotics(dactinomycin (actinomycin D), daunorubicin, doxorubicin, andidarubicin), anthracyclines, mitoxantrone, bleomycins, plicamycin(mithramycin), and mitomycin, enzymes (L-asparaginase, whichsystemically metabolizes L-asparagine and deprives cells that do nothave the capacity to synthesize their own asparagine); antiplateletagents; antiproliferative/antimitotic alkylating agents such as nitrogenmustards (mechlorethamine, cyclophosphamide, and analogs, melphalan,chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine andthiotepa), alkyl sulfonates (busulfan), nitrosoureas (carmustine (BCNU)and analogs, streptozocin), trazenes-dacarbazinine (DTIC);antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate), pyrimidine analogs (fluorouracil, floxuridine, andcytarabine), purine analogs and related inhibitors (mercaptopurine,thioguanine, pentostatin, and 2-chlorodeoxyadenosine); aromataseinhibitors (anastrozole, exemestane, and letrozole); and platinumcoordination complexes (cisplatin, carboplatin), procarbazine,hydroxyurea, mitotane, aminoglutethimide; histone deacetylase (HDAC)inhibitors (trichostatin, sodium butyrate, apicidan, suberoyl anilidehydroamic acid); hormones (i.e., estrogen) and hormone agonists such asleutinizing hormone releasing hormone (LHRH) agonists (goserelin,leuprolide, and triptorelin). Other chemotherapeutic agents may includelenalidomide, mechlorethamine, camptothecin, ifosfamide, tamoxifen,raloxifene, gemcitabine, navelbine, or any analog or derivative variantof the foregoing.

In some embodiments, the present invention relates to a method oftreating cancer (e.g., multiple myeloma or solid tumor) in a subject inneed of treatment. The method typically comprises administering atherapeutically effective amount of a pharmaceutical liposomalcomposition of the present invention (e.g., comprising carfilzomib), andmay further comprise simultaneous, sequential, or separateadministration of a therapeutically effective amount of achemotherapeutic agent.

In certain embodiments of the present invention, a liposomal compositiondescribed herein is used in a combination treatment that includes acytokine Cytokines include, but are not limited to, Interferon-γ,Interferon-α, and Interferon-β; Interleukins 1-8, 10, and 12;Granulocyte Monocyte Colony-Stimulating Factor (GM-CSF); TNF-α andTNF-β; and TGF-β.

Embodiments of the present invention include combination treatmentsincorporating a liposomal composition described herein and a steroid.Suitable steroids may include, but are not limited to,21-acetoxypregnenolone, alclometasone, algestone, amcinonide,beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol,clocortolone, cloprednol, corticosterone, cortisone, cortivazol,deflazacort, desonide, desoximetasone, dexamethasone, diflorasone,diflucortolone, difuprednate, enoxolone, fluazacort, flucloronide,flumethasone, flunisolide, fluocinolone acetonide, fluocinonide,fluocortin butyl, fluocortolone, fluorometholone, fluperolone acetate,fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasonepropionate, formocortal, halcinonide, halobetasol propionate,halometasone, hydrocortisone, loteprednol etabonate, mazipredone,medrysone, meprednisone, methylprednisolone, mometasone furoate,paramethasone, prednicarbate, prednisolone, prednisolone25-diethylaminoacetate, prednisolone sodium phosphate, prednisone,prednival, prednylidene, rimexolone, tixocortol, triamcinolone,triamcinolone acetonide, triamcinolone benetonide, triamcinolonehexacetonide, and salts and/or derivatives thereof.

In certain embodiments of the present invention, a liposomal compositiondescribed herein is part of a combination treatment that includes animmunotherapeutic agent. Suitable immunotherapeutic agents may include,but are not limited to, MDR modulators (verapamil, valspordar,biricodar, tariquidar, laniquidar), cyclosporine, thalidomide, andmonoclonal antibodies. The monoclonal antibodies can be either naked orconjugated such as rituximab, tositumomab, alemtuzumab, epratuzumab,ibritumomab tiuxetan, gemtuzumab ozogamicin, bevacizumab, cetuximab,erlotinib, and trastuzumab.

Experiments performed in support of the present invention demonstratedthat liposomal compositions of the present invention provided increasedmaximum tolerated dose (MTD) relative to a non-liposomal compositioncomprising peptide epoxyketone compound. For example, in mice, a firstliposomal composition resulted in a 2.5-fold increase in the MTD, and asecond liposomal composition resulted in a 50% increase. In rats, bothliposomal compositions resulted in increases in tolerability (Example4). Biodistribution, as measured by proteasome inhibition in blood andtissues, was similar across the various compositions (Example 5, Example11). Further, the liposomal peptide epoxyketone compound compositions ofthe present invention provided about 3 to 5 and 7-fold increasedexposure (AUC) in mice and rats, respectively, compared to anon-liposomal composition comprising peptide epoxyketone compound. Thisincreased exposure was the result of a decrease in plasma clearance(Example 6, Example 11).

Further, liposomal compositions comprising a peptide epoxyketonecompound entrapped in the liposomes' aqueous core demonstrated enhancedtolerability by increasing the maximum tolerated dose (MTD) ofcarfilzomib in mice by 50%, from 10 mg/kg to 15 mg/kg as compared to theinjectable, non-liposomal, SBE-CD formulation. These results indicatethat liposomal compositions comprising a peptide epoxyketone compoundentrapped in the liposomes' aqueous core release carfilzomib over alonger period of time with a lower maximum plasma concentration (Cmax)relative to the injectable, non-liposomal, SBE-CD formulation.

The liposomal compositions comprising liposomes having a peptideepoxyketone compound entrapped in their aqueous core also resulted indelayed proteasome recovery at 24 hours in some mouse tissues whereasthe current drug product (i.e., injectable, non-liposomal, CFZ SBE-CD)resulted in recovery from proteasome inhibition by 24 hours post-dose(Example 8, FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D). These results supportthat the liposomal compositions provide long-term exposure of peptideepoxyketone compounds.

In addition, when delivered in the non-liposomal CFZ SBE-CD formulation,the plasma concentration of carfilzomib declined rapidly and was notdetectable after 1 hour post-dose (Example 9, FIG. 4). When carfilzomibwas delivered as liposomal compositions comprising a peptide epoxyketonecompound entrapped in the liposomes' aqueous core, systemic exposure wasextended with detectable total drug (both encapsulated and released) forup to 24 hours post-dose (Example 9, FIG. 4). These data demonstratethat the liposomal compositions comprising a peptide epoxyketonecompound entrapped in the liposomes' aqueous core resulted insignificantly greater exposure and longer circulation relative to aninjectable, non-liposomal, peptide epoxyketone compound formulation.

Also, liposomal compositions of the present invention maintain efficacyat a reduced dosing frequency relative to a non-liposomal formulation(Example 11).

Accordingly, the data in the Examples demonstrate that liposomalcompositions of the present invention resulted in prolonged exposurewithout affecting biodistribution. Tolerability of the peptideepoxyketone compound was also enhanced in animals, likely due to reducedexposure to high concentrations of free drug. The liposomal compositionscomprising peptide epoxyketone compounds of the present inventionprovide the following improvements relative to the current injectable,non-liposomal, CFZ SBE-CD formulation: improve the pharmacodynamicprofile of peptide epoxyketone compounds by delaying proteasomerecovery; improve the pharmacokinetic profile by decreasing clearanceand extending plasma half-life; improve the safety profile of peptideepoxyketone compounds (i.e. tolerability); and allow reduced dosingfrequency.

Experimental

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how topractice the present invention, and are not intended to limit the scopeof what the inventors regard as the invention. Efforts have been made toensure accuracy with respect to numbers used (e.g., amounts,concentrations, percent changes, etc.) but some experimental errors anddeviations should be accounted for. Unless indicated otherwise,temperature is in degrees Centigrade and pressure is at or nearatmospheric.

The compositions used to practice the methods of the present inventionmeet the specifications for content and purity required ofpharmaceutical products.

1.0 MATERIALS AND METHODS

Particle Size Reduction of Liposomal Suspension

Particle size reduction or deagglomeration of the rehydrated liposomalsuspension can be carried out either by sonication (20 to 60 minutes) orby high-pressure homogenization/microfluidizer (up to 30,000 psi).

Content Determination by HPLC Assay

The liposomal suspension can be mixed with an organic solvent, forexample, methanol, to dissolve lipids and free the carfilzomib. Thesolution can be filtered through a 0.2 μm PTFE filter prior to HPLCanalysis.

Carfilzomib content can be determined by a gradient HPLC assay accordingto the method in Table 1 using sodium perchlorate buffer, 0.1M, pH 3.1and acetonitrile (50:50 v/v).

TABLE 1 Column: Phenomenex Gemini ™ C18, 150 × 4.6 mm, 5 μm particlesize Column Temperature: 30 ± 2° C. Autosampler Temperature: 5 ± 3° C.Detection Wavelength: 220 nm Flow Rate: 1.5 mL/min Injection Volume: 10μL Total Run Time: 17 min

Liposome Morphology

The vesicle size, shape and surface morphology of the liposomalcomposition can be determined by scanning electron microscopy (SEM) andtransmission electron microscopy (TEM).

Liposome/Carfilzomib Solid State

Polarized light microscopy, differential scanning calorimetry (DSC),X-ray diffraction (XRD), and freeze fracture electron microscopy can beused to elucidate the phase behavior of the vesicles.

Liposome Size and Distribution

Polarized light microscopy, dynamic light scattering, and TEM can beused to determine the size and size distribution range of the liposomesof the liposomal composition.

Determination of Free Drug

Because carfilzomib has extremely low aqueous solubility, the amount ofentrapped drug versus free drug can be qualitatively determined based onpolarized light microscope. Free drug precipitates in the aqueousmedium, due to its extremely low aqueous solubility, and theprecipitated material can be seen using a polarized light microscope.

Sulfobutyl ether-β-cyclodextrins

Sulfobutyl ether-β-cyclodextrins (SBE-CD), for example, CAPTISOL®, canbe synthetically produced and/or are commercially available, for examplefrom Ligand Pharmaceuticals, Inc., La Jolla, Calif.

2.0 EXAMPLES Example 1 Preparation of Molecularly Dispersed Carfilzomibin Thin Lipid Film

To make liposomal carfilzomib, the following materials in the indicatedratios were added to a suitably sized round bottom flask: drug to totallipid weight ratio of 1:19 to 1:2.33. Total lipids typically comprisethe lipids EPC, HSPC, DSPC, DPPC, DSPE, and/or sphingomyelin (SPH)alone, or with cholesterol. If cholesterol is added, the lipid tocholesterol weight ratio (lipid:cholesterol) is from 0.9:0.1 to 0.5:0.5.An appropriate volume of phosphate buffer saline was used to rehydratethe lipid film to give a target carfilzomib concentration of 1 and 2mg/mL, respectively.

To make PEGylated liposomal carfilzomib, the following materials in theindicated ratios were added to a suitably sized round bottom flask: drugto total lipid weight ratio of 1:19 to 1:2.33. Total lipids typicallycomprise the lipids EPC, HSPC, DSPC, DPPC, DSPE, and/or sphingomyelinwith PEG-modified lipids (e.g., PEG-modified phospholipids) in weightratio (lipids:PEG-modified lipids) of from 0.9:0.1 to 0.75:0.25, or whencholesterol is added the lipid to PEG-modified lipid to cholesterolweight ratio (lipids:PEG-modified lipids:cholesterol) of from0.83:0.083:0.083 to 0.57:0.14:0.29. An appropriate volume of phosphatebuffer saline was used to rehydrate the lipid film to give a targetcarfilzomib concentration of 2 mg/mL.

To dissolve the lipids and carfilzomib, an appropriate volume of organicsolvent (e.g., cloroform:MeOH (60:40 or 50:50 v/v)), enough to achievebetween 10-20 mg/mL dissolved lipid, was added to the flask. The flaskwas attached to a rotary evaporator spinning at 50-100 rpm and immersedin a water bath set above the highest gel-liquid crystal phasetransition (Tc) temperature of the lipids used. Although the Tc for eggphosphatidylcholine is below room temperature (−15° C. to −7° C.), thetemperature bath used for EPC was approximately 45° C. to 50° C. ForDSPC, DPPC, and mPEG-DSPE, the water bath temperature should be setgreater than 55° C., 41° C., and 50° C., respectively.

The flask was allowed to rotate in the water bath for approximately 1minute to equilibrate. A slow vacuum was pulled, to as low as <10 Torr,to obtain a thin dry film on the walls of the flask withoutprecipitation. To remove any residual solvent, the flask was subjectedto high vacuum at room temperature for a few hours or overnight. Table 2presents nominal concentrations of the components of exemplary liposomalcarfilzomib compositions (liposomal carfilzomib compositions, L-CFZ;pegylated liposomal carfilzomib compositions, pL-CFZ), as well ascontrol compositions (i.e., “empty” liposomes).

TABLE 2 mPEG- Choles- Composition Composition CFZ EPC DSPE terolDesignation Name (mg/mL) (mg/mL) (mg/mL) (mg/mL) A Empty 0 12.5 0 0Liposomes B 1 mg/mL 1 12.5 0 0 Liposomal CFZ C 2 mg/mL 2 12.5 0 0Liposomal CFZ D Empty PEG- 0 12.5 1.3 0 Liposomes E 2 mg/mL 2 12.5 1.3 0PEG- Liposomal CFZ F Empty PEG- 0 12.5 1.3 3.1 Liposomes w/Cholesterol G2 mg/mL 2 12.5 1.3 3.1 PEG- Liposomal CFZ w/Cholesterol

Table 3 presents example ranges of components to be used in liposomalcarfilzomib compositions of the present invention, as well as controlcompositions (i.e., “empty” liposomes). In the table, where EPC islisted, additional lipids can also be added for example, HSPC, DSPC,DPPC, DSPE, and/or sphingomyelin.

TABLE 3 Composition Type Weight ratio Empty Liposomes = Empty L CFZ toEPC w/w ratio 0:1 Liposomal CFZ = L-CFZ CFZ to EPC w/w ratio from 1:19to 1:2.33 Empty Liposomes w/Cholesterol = CFZ to Total Lipid¹ w/w ratio0:1 Empty L Chol where EPC to Cholesterol w/w ratio is from 0.9:0.1 to0.5:0.5 Liposomal CFZ w/Cholesterol = CFZ to Total Lipid¹ w/w ratio fromL-CFZ Chol 1:19 to 1:2.33 where EPC to Cholesterol w/w ratio is from0.9:0.1 to 0.5:0.5 Empty PEGylated Liposomes = CFZ to Total Lipid² w/wratio 0:1 Empty pL where EPC to mPEG w/w ratio is from 0.9:0.1 to0.75:0.25 PEGylated Liposomal CFZ = CFZ to Total Lipid² w/w ratio frompL-CFZ 1:19 to 1:2.33 where EPC to mPEG w/w ratio is from 0.9:0.1 to0.75:0.25 Empty PEGylated Liposomes CFZ to Total Lipid³ w/w ratio 0:1w/Cholesterol = Empty pL Chol where EPC to mPEG to Cholesterol w/w ratiois from 0.83:0.083:0.083 to 0.57:0.14:0.29 PEGylated Liposomal CFZ w CFZto Total Lipid³ w/w ratio 1:19 to Cholesterol = pL-CFZ Chol 1:2.33 whereEPC to mPEG to Cholesterol w/w ratio is from 0.83:0.083:0.083 to0.57:0.14:0.29 ¹Total Lipid = EPC + Cholesterol; ²Total Lipid = EPC +mPEG; ³Total Lipid = = EPC + mPEG + Cholesterol

Example 2 Lipid Hydration

The thin-filmed, round-bottom flask was immersed in a water bath setabove the highest gel-liquid crystal phase transition. When EPC wasused, rehydration occurred at room temperature. For DSPC, DPPC, andmPEG-DSPE, the water bath temperature should be set greater than 55° C.,41° C., and 50° C., respectively. An appropriate volume of phosphatebuffered saline, pH 7.2, or water for injection was added to the lipidfilm to achieve the desired target carfilzomib concentration or dose.The flask was mildly agitated or shaken with intermittent vortexing, asneeded, and sonicated in water bath at the appropriate Tc temperaturefor 1 to 2 minutes to facilitate complete hydration from flask walls.

After the film was dispersed, the mixture was transferred to a vial andsonicated for an additional 20 to 40 minutes in a water bath above theTc to size the liposomes. When EPC was used, the temperature of thewater bath in the sonicator was kept near room temperature. Uponhydration the lipid suspension appeared as a slightly hazy or milkysolution.

FIG. 1 sets forth the composition of exemplary liposomal carfilzomibcompositions of the present invention, as well as control compositions(i.e., “empty” liposomes) used in the studies described below.

Example 3 Characterization of Liposomes

Particle size reduction and/or deagglomeration of the rehydratedliposomal suspension was carried out by sonication (20 to 60 minutes).

The CFZ content of exemplary liposomal carfilzomib compositions wasdetermined by HPLC as described above. The liposomal compositions wereeach diluted in methanol to dissolve lipids and carfilzomib. Thesolution was filtered through a 0.2μm PTFE filter prior to HPLCanalysis. The percent difference between the theoretical andexperimental liposomal drug concentrations for the prepared lots(FIG. 1) were typically 2% or less (except for one of the first lotswhich had a 12% difference). The results of the HPLC analysis arepresented in FIG. 1.

Based on polarized light microscope it was qualitatively determined thatnearly all of the drug was entrapped in the liposomes as any free drugwould precipitate in the aqueous medium due to its extremely low aqueoussolubility of <1 μg/mL and any precipitated material could be easilyobserved under the polarized light microscope.

Example 4 Tolerability of Liposomal Carfilzomib

The tolerability of carfilzomib incorporated in either liposomal (L-CFZ)or pegylated liposomal (pL-CFZ) carfilzomib compositions was evaluatedin both mice and rats above the maximum tolerated dose (MTD) achievedusing an injectable composition of carfilzomib formulated in 10%sulfobutylether beta cyclodextrin (SBE-β-CD, also referred to as CFZSBE-CD), 10 mM Citrate, pH 3.5 (see, e.g., U.S. Patent Publication Nos.2011/0236428).

Liposomal compositions prepared in Example 2 and characterized inExample 3 were rehydrated with an appropriate volume of aqueous mediumto achieve a target carfilzomib concentration in the range ofapproximately 1 to 2 mg/ml (see composition data in FIG. 1). Toxicityfor both L-CFZ and pL-CFZ compositions were tested in mice (Table 4).Toxicity for L-CFZ was tested in rats (Table 5).

Female BALB/c mice (7-8 week old; 5/cohort) were dosed intravenously asfollows: 15 mg/kg CFZ SBE-CD (7.5 mL/kg); 10 mg/kg L-CFZ (5 mL/kg); 10mg/kg pL-CFZ (5 mL/kg); 15 mg/kg L-CFZ (7.5 mL/kg); 15 mg/kg pL-CFZ (7.5mL/kg); 20 mg/kg L-CFZ (10 mL/kg); 20 mg/kg pL-CFZ (10 mL/kg); 25 mg/kgL-CFZ (12.5 mL/kg); 25 mg/kg pL-CFZ (12.5 mL/kg); 30 mg/kg L-CFZ (15mL/kg); 35 mg/kg L-CFZ (17.5 mL/kg); or empty liposome (15 mL/kg).).Survival was then monitored over a seven day period. The survival ratesof mice, the liposomal compositions, and dosing used for treatment wereas shown in Table 4.

TABLE 4 Overall Mortality Dose (No. Group Composition # (Dose Volume)(mg/kg) dead/total) CFZ alone 2 m g/mL SBE-CD Composition 15 5/5 (10mg/kg MTD) (150 μL) Empty Composition A (300 μL) 0 0/5 Liposome L-CFZComposition B (200 μL) 10 0/5 L-CFZ Composition C (150 μL) 15 0/5 L-CFZComposition C (200 μL) 20 0/5 L-CFZ Composition C (250 μL) 25 0/5 L-CFZComposition C (300 μL) 30 1/5 L-CFZ Composition C (350 μL) 35 4/5 EmptyPEG Composition D (300 μL) 0 0/5 Liposome pL-CFZ Composition E (100 μL)10 0/5 pL-CFZ Composition E (150 μL) 15 0/5 pL-CFZ Composition E (200μL) 20 5/5 pL-CFZ Composition E (250 μL) 25 5/5

Male Sprague Dawley rats (5/cohort) weighing approximately 250-300 gramswere dosed intravenously with the following: 8 mg/kg CFZ (SBE-CDcomposition) (5 mL/kg); 8 mg/kg L-CFZ (4 mL/kg); 10 mg/kg L-CFZ (5mL/kg); 12.5 mg/kg L-CFZ (6 mL/kg); or empty liposome (5 mL/kg).Survival was then monitored over a seven day period. The survival ratesof dosed rats were as shown in Table 5.

TABLE 5 Overall Mortality Dose (No. Group Composition # (mL/kg) (mg/kg)dead/total) CFZ (7 mg/kg SBE-CD Composition 8 2/5 MTD) (5 mL/kg) EmptyComposition A (5 mL/kg) 0 0/5 Liposome L-CFZ Composition C (4 mL/kg) 80/5 L-CFZ Composition C (5 mL/kg) 10 0/5 L-CFZ Composition C (6 mL/kg)12.5 3/5

Liposomal carfilzomib significantly enhanced tolerability by increasingthe maximum tolerated dose (MTD) of carfilzomib in mice by approximately2.5 fold for the liposomal carfilzomib compositions and by 50% withPEGylated liposomal carfilzomib compositions compared to SBE-CD basedcarfilzomib composition. Only a slight increase in the MTD in rats wasobserved with liposomal carfilzomib L-CFZ (10 mg/kg) compared tocarfilzomib (7 mg/kg) formulated in SBE-CD.

These data demonstrate that liposomal compositions comprising peptideepoxyketone compounds significantly enhanced tolerability by increasingthe maximum tolerated dose (MTD) of a peptide epoxyketone compound.

Example 5 Pharmacodynamic Response of CFZ Liposomal Compositions

The pharmacodynamic response of carfilzomib formulated in SBE-CD (CFZSBE-CD) (using an injectable composition of carfilzomib formulated in10% sulfobutylether beta cyclodextrin (SBE-CD), 10 mM Citrate, pH 3.5(see, e.g., U.S. Patent Publication Nos. 2011/0236428)), empty pL(Composition D, FIG.1), liposomes comprising CFZ (L-CFZ, Composition C,FIG. 1), and pegylated liposomes comprising (pL-CFZ, Composition E,FIG. 1) was evaluated in BALB/C mice following a single intravenousbolus administration.

The mice (three mice per time point) were administered a dose of 10mg/kg in a dose volume of 5 mL/kg. Blood samples and tissues forpharmacodynamic testing were taken at 1, 8, and 24 hours afteradministration of each composition. The pharmacodynamic response wasdetermined by measurement of proteasome activity in whole blood(primarily erythrocytes) (see FIG. 2A), adrenal (see FIG. 2B), liver(see FIG. 2C), and heart (see FIG. 2D), using a fluorogenic substrate(LLVY-AMC; as described by Lightcap E S, McCormack T A, Pien C S, etal., Clin. Chem. 46:673-683 (2000)) to quantitate the chymotrypsin-likeactivity of the proteasome. All samples were normalized to the CFZvehicle, and the vehicle time point was 1 hour post dose. Three tissuesamples were evaluated per time point for each tissue from each mouse.

A single intravenous dose of 10 mg/kg resulted in rapid proteasomeinhibition of >80% within 1 hour in whole blood and all tissues. Similarand complete recovery from proteasome inhibition was observed 24 hourspost-dose in all tissues tested except for the blood and heart andoccurred with at t1/2 of 8-24 hours for all compositions. The slowerrecovery observed in the heart with both the liposomes and pegylatedliposomes suggest that the heart tissue may act as a depot. As expected,there was no recovery of proteasome activity in blood due to theirreversible binding of carfilzomib and the lack of the erythrocytes tosynthesize new proteasome.

These observations indicate that inhibition of proteasome activity inwhole blood and tissues is rapid, similar across compositions andcorrelates with rapid absorption. The liposomal compositions did notadversely affect biodistribution of CFZ.

Example 6 Circulation Half-Life of Liposomal CFZ

Circulation half-life of liposomal CFZ was evaluated in 7-8 week oldfemale BALB/c mice (3/timepoint) following a single i.v. injection ofeither 5 mg/kg CFZ formulated in 10% sulfobutylether beta cyclodextrin,10 mM Citrate, pH 3.5 or 15 mg/kg of liposomal carfilzomib compositions.

When CFZ was delivered in the composition containing SBE-CD at 5 mg/kg,plasma concentration rapidly declines with time and drops to below thelimit of quantitation (BLOQ; limit of quantitation—LOQ) after 60 minutes(Table 6). The plasma half-life (t1/2) was about 20 minutes.

TABLE 6 Plasma Levels of CFZ Using SBE-CD Composition I.V. Bolus 5 mg/kgin BALB/c Mice Plasma Conc. (uM) Time (min) Mean STD 0 0.0 0 2 10.3790.844 5 1.732 0.431 10 0.310 0.064 20 0.176 0.040 30 0.061 0.011 600.042 0.032 LOQ = 1 ng/mL (MW = 719.4)

When delivered in liposomal compositions at 15 mg/kg (using L-CFZ,Composition-C, or pL-CFZ-Chol, Composition G, (FIG. 1) with a dosevolume of 150 μL), detectable CFZ was observed at 6 hours post-dosing(Table 7).

TABLE 7 Mean Plasma Levels of Liposomal CFZ IV bolus at 15 mg/kg inBALB/c Mice Plasma Conc. (uM) Plasma Conc. (uM) Liposomal CFZ PEGylatedLiposomal CFZ Time (min) Mean STD Mean STD predose BLOQ BLOQ BLOQ BLOQ 2102 21 79.0 22.7 5 51.4 9.1 42.5 8.4 10 22.2 6.2 9.81 3.48 30 1.26 0.480.183 0.045 60 0.143 0.018 0.0537 0.0200 120 0.0424 0.0134 0.0111 0.0010240 0.0125 0.0010 0.0152 0.0070 360 0.0129 0.0023 0.0122 0.0100

The t1/2 was 140 and 201 minutes, respectively, for liposomal CFZ andpegylated liposomal CFZ compositions, respectively. The data forliposomal CFZ compositions versus CFZ SBE-CD, clearly demonstrate theability of liposome to significantly enhance the circulation half-lifeof peptide epoxyketone compounds.

Circulation half-life of liposomal CFZ was also evaluated in maleSprag-Dawley rats (3/timepoint) weighing approximately 250-300 gramsfollowing a single i.v. injection of 8 mg/kg CFZ formulated in 10%sulfobutylether beta cyclodextrin, 10 mM Citrate, pH 3.5, or 8 mg/kgliposomal CFZ. Similar to mice, a rapid decline in plasma concentrationwas observed in rats when CFZ SBE-CD was delivered (Table 8), with aplasma t1/2 of 17 minutes. When CFZ was delivered in liposomalcompositions (L-CFZ, Composition C, FIG. 1, with a dose volume of 4mL/kg) at the same dose level, detectable CFZ was observed at 4 hourspost-dosing (Table 8), with a t1/2 of about 50 minutes.

TABLE 8 Plasma Levels of CFZ Using SBE-CD Composition or Liposomal CFZ(IV bolus at 8 mg/kg) in Rats SBE-CD Composition Liposomal-CFZ PlasmaConc. (uM) Time (min) Mean STD Mean STD predose BLOQ BLOQ BLOQ BLOQ 0.142.9 4.4 ND ND 1 3.93 0.36 ND ND 2 1.90 0.26 36.9 4.8 5 0.651 0.115 21.01.8 15 0.0505 0.0030 0.583 0.072 30 0.0189 0.0030 0.139 0.050 60 0.00720.0020 0.058 0.029 120 ND ND 0.012 0.003 240 ND ND 0.004 0.001 420 ND NDBLOQ BLOQ

The data presented in Table 9 and Table 10 demonstrate that, compared toCFZ SBE-CD, the exposure (AUC) to liposomal CFZ compositions (L-CFZComposition C, FIG. 1) and pegylated liposomal CFZ compositions (pL-CFZComposition E, FIG. 1), was increased about 5 to 7 and 20-fold in miceand rats, respectively.

TABLE 9 Mean AUC Levels of CFZ Using SBE-CD Composition or Liposomal CFZ(IV bolus at 15 mg/kg) in Mice Relative Increase AUCinf AUC-_(liposome)/Species Dose (mg/kg) Composition (min*μmol/L) AUC-_(SBE-CD) Mouse 15SBE-CD 130.5 — Mouse 15 L-CFZ-C 942.8 7 Mouse 15 pL-CFZ-E 623.3 5

TABLE 10 Mean AUC Levels of CFZ Using SBE-CD Composition or LiposomalCFZ (IV bolus at 8 mg/kg) in Rats Relative Increase AUCinfAUC-_(liposome)/ Species Dose (mg/kg) Composition (min*μmol/L)AUC-_(SBE-CD) Rat 8 SBE-CD 14.5 — Rat 8 L-CFZ-C 297.7 20

These data demonstrate the extended duration of exposure to peptideepoxyketone compounds in liposomal composition versus the non-liposomalSBE-CD composition.

Example 7 Preparation of Thin Lipid Film and use in Preparing LiposomesComprising an Aqueous Core Loaded with Peptide Epoxyketone CompoundsComplexed with SBE-CD

To make the PEGylated liposomal film, the following materials at theirindicated ratios were added to a suitably sized round bottom flask.Total lipids typically comprise the lipids EPC, DSPC, DPPC, and/or SPHwith PEG-modified lipids (e.g., PEG-modified phospholipids) in weightratio (lipids:PEG-modified lipids) of from 0.9:0.1 to 0.75:0.25, or whencholesterol is added the lipid to PEG-modified lipid to cholesterolweight ratio (lipid:PEG-modified lipid:cholesterol) of from0.83:0.083:0.083 to 0.57:0.14:0.29.

To dissolve the lipids, an appropriate volume of organic solvent (e.g.,cloroform:MeOH (60:40 v/v)), enough to achieve between 10-20 mg/mLdissolved lipid, was added to the flask. The flask was attached to arotary evaporator spinning at 100 rpm and immersed in a water bath setabove the highest gel-liquid crystal phase transition (Tc) temperatureof the lipids used. Although the Tc for egg phosphatidylcholine is belowroom temperature (−15° C. to −7° C.), the temperature bath used for EPCwas approximately 45° C. to 50° C. For DSPC, DPPC, and mPEG-DSPE, thewater bath temperature should be set greater than 55° C., 41° C., and50° C., respectively. If there is no phase transition temperature, thewater bath temperature is set between 35-45° C.

The flask was allowed to rotate in the water bath for approximately 1minute to equilibrate. A slow vacuum was pulled, to as low as <10 Torr,to obtain a thin dry film on the walls of the flask withoutprecipitation (typically for about 30 minutes). To remove any residualsolvent, the flask was subjected to high vacuum at room temperature fora few hours or overnight.

Carfilzomib (CFZ) was solubilized in an aqueous solution by complexationwith sulfobutylether beta cyclodextrin (SBE-CD). Excess carfilzomib wasadded to an aqueous solution of 20% SBE-CD and 20 mM citric acid. Thesolution pH was adjusted to approximately pH 2.5 with 1N HCl, if neededto solubilize CFZ. The mixture was sonicated for approximately 10minutes and stirred using a magnetic stir bar for not less than an hourprior to filtration through a 0.2 μm filter to remove excess undissolveddrug. After filtration the solution pH was adjusted to between pH 3.5and 5. This aqueous solution of CFZ complexed with SBE-CD was used torehydrate the thin lipid film.

Once the vesicles were rehydrated the unencapsulated free drug wasremoved by centrifugation at 31000 rpm for 30 minutes and washing withPBS or by being dialyzed using a membrane with a MWCO of 8-10 kD in PBSfor up to 48 hours.

Table 11 presents nominal concentrations of the components of exemplaryliposomal compositions comprising liposomes comprising an aqueous coreloaded with CFZ complexed with SBE-CD.

TABLE 11 Composition Drug Designation Lipid composition content * apL12.5 mg/mL EPC, 3.3 mg/mL cholesterol, 0.9 mg/mL 1.3 mg/mL mPEG-DSPEapL-9 25 mg/mL EPC, 6.3 mg/mL cholesterol, 1.3 mg/mL 2.5 mg/mL mPEG-DSPEapL-11 (for 15 12.5 mg/mL egg SPH, 3.3 mg/mL cholesterol, 0.8 mg/mLmg/kg dosing) 1.3 mg/mL mPEG-DSPE apL-11 (for 5 12.5 mg/mL egg SPH, 3.3mg/mL cholesterol, 0.2 mg/mL mg/kg dosing) 1.3 mg/mL mPEG-DSPE * Totaldrug content may include unencapsulated drug that was not removed duringprocessing.

Example 8 Pharmacodynamic Response of CFZ Liposomal CompositionsComprising Liposome Comprising an Aqueous Core Loaded with PeptideEpoxyketone Compounds Complexed with SBE-CD

The pharmacodynamic response of liposomal compositions comprisingliposomes comprising an aqueous core loaded with CFZ complexed withSBE-CD was evaluated in BALB/C mice following a single intravenous bolusadministration.

The pharmacodynamic response of injectable carfilzomib formulated inSBE-CD (non-liposomal; see, e.g., U.S. Patent Publication Nos.2011/0236428) or liposomal compositions comprising liposomes comprisingan aqueous core loaded with CFZ complexed with SBE-CD was evaluated inBALB/C mice (apL-11 (for 15 mg/kg dosing), Example 7; and apL-11 (for 5mg/kg dosing), Example 7) following a single intravenous bolusadministration. The mice (three mice per time point) were administered adose of 5 or 10 mg/kg of non-liposomal carfilzomib or 5 or 15 mg/kg ofliposomal carfilzomib as a solution in a dose volume of 5 mL/kg. Bloodsamples and tissues for pharmacodynamic testing were taken at 0, 1, 8,and 14 hours after administration of the non-liposomal composition at 10mg/kg; 0, 1, 4, 6, 8 and 24 hours after administration of thenon-liposomal composition at 5 mg/kg; and 0, 1, 4, 6, and 24 hours afteradministration of the liposomal compositions at 5 mg/kg and 15 mg/kg.Three tissue samples were evaluated per time point for each tissue fromeach mouse. The pharmacodynamic response was determined by measurementof proteasome activity in whole blood (primarily erythrocytes), adrenal,liver, and heart using a fluorogenic substrate (LLVY-AMC; as describedby Lightcap E S, McCormack T A, Pien C S, et al., Clin. Chem. 46:673-683(2000)) to quantitate the chymotrypsin-like activity of the proteasome.All samples were normalized to the corresponding vehicle without CFZ,and the vehicle sample time point measurement was 1 hour post dose.

A single dose of injectable carfilzomib formulated in SBE-CD(non-liposomal) at either 5 or 10 mg/kg or liposomal compositionscomprising liposomes comprising an aqueous core loaded with CFZcomplexed with SBE-CD (apL-11) at either 5 or 15 mg/kg resulted in arapid inhibition of proteasome activity within 1 hour in whole blood andall other tissues. Greater inhibition of proteasome activity wasobserved at the 15 mg/kg dose which resulted in >80% inhibition ofproteasome activity at 1 hour in all tissue: whole blood (primarilyerythrocytes) (see FIG. 3A), heart (see FIG. 3B), liver (see FIG. 3C),and adrenal (see FIG. 3D). Similar and near complete recovery fromproteasome inhibition was observed 24 hours post-dose in all tissuestested except for the blood and heart and occurred with at t1/2 of 6-24hours for both the 5 and 10 mg/kg dose levels of injectable carfilzomibformulated in SBE-CD (non-liposomal). Delayed recovery of proteasomeactivity in the adrenals was observed with liposomal compositionscomprising liposomes comprising an aqueous core loaded with CFZcomplexed with SBE-CD at both 5 and 15 mg/kg; this result suggests longterm exposure of CFZ. As expected, there was no recovery of proteasomeactivity in blood due to the irreversible binding of carfilzomib and thelack of the erythrocytes to synthesize new proteasome.

These observations indicate that inhibition of proteasome activity inwhole blood and tissues was rapid and similar across between thenon-liposomal formulation and liposomal compositions comprising peptideepoxyketone compounds. Further, the delay in the recovery of proteasomeactivity in the adrenals with liposomal compositions comprisingliposomes comprising an aqueous core loaded with peptide epoxyketonecompound complexed with SBE-CD suggests extended exposure with theliposomal composition versus the non-liposomal composition. Further, theliposomal compositions comprising peptide epoxyketone compounds did notadversely affect biodistribution of the peptide epoxyketone compounds.

Example 9 Circulation Half-Life of Liposomal CFZ

Circulation half-life of liposomal CFZ was evaluated in 7-8 week oldfemale BALB/c mice (3/timepoint) following a single i.v. injection ofthe following: 5 mg/kg CFZ formulated in 10% sulfobutylether betacyclodextrin (CAPTISOL®), 10 mM Citrate, pH 3.5 (CFZ SBE-CD;non-liposomal); 5 mg/kg of liposomal carfilzomib compositions apL-11(Example 7; apL-11 (for 5 mg/kg dosing)) and 15 mg/kg of liposomalcarfilzomib compositions apL-11 (Example 7; apL-11 (for 15 mg/kgdosing)).

As shown in FIG. 4, the plasma concentration of injectable carfilzomibSBE-CD composition (non-liposomal) declined rapidly followingintravenous, bolus administration due to rapid and extensive metabolism(FIG. 4: line with open squares containing an X corresponds toadministration of 5 mg/kg of an injectable carfilzomib SBE-CDcomposition (non-liposomal)). The half-life of carfilzomib dosed at 5mg/kg was about 20 minutes and carfilzomib was not detectable after 1hour post-dose.

When delivered in liposomal compositions, the duration of exposure tocarfilzomib was greatly extended (FIG. 4, line with solid squarescorrespond to administration of 5 mg/kg of apL11 (Example 7), apegylated liposomal composition of carfilzomib wherein the aqueous coreof the pegylated liposomes comprises carfilzomib and SBE-CD; line withsolid circles correspond to administration of 15 mg/kg of apL11 (Example7), a pegylated liposomal composition of carfilzomib wherein the aqueouscore of the pegylated liposomes comprises carfilzomib and SBE-CD). Totaldrug (encapsulated and released) was detectable for up to 24 hourspost-dose. This is consistent with the observed delay in proteasomerecovery in tissues.

The data for liposomal CFZ compositions versus CFZ SBE-CD(non-liposomal), demonstrate the ability of liposomal compositions tosignificantly enhance the circulation half-life of peptide epoxyketonecompounds. Further, the data show the ability to provide extendedduration of exposure to peptide epoxyketone compounds in liposomalcompositions versus the non-liposomal SBE-CD composition.

Example 10 Preparation of Liposomes Comprising an Aqueous Core Loadedwith Peptide Epoxyketone Using pH Control and an Ethanol InjectionMethod

To make the liposomal compositions using an ethanol injection method,the following materials at their indicated ratios were used. Totallipids typically comprise the lipids EPC, DSPC, DPPC, and/or SPH, andcan further comprise PEG-modified lipids (e.g., PEG-modifiedphospholipids) and/or cholesterol in weight ratio (lipids:PEG-modifiedlipids or lipids:cholesterol) of 0.9:0.1 to 0.75:0.25, or whencholesterol is added the lipid to PEG-modified lipid to cholesterolweight ratio (lipid:PEG-modified lipid:cholesterol) can be from0.83:0.083:0.083 to 0.57:0.14:0.29.

Other materials used in the ethanol injection method include thefollowing: absolute Ethanol; 1N HCl; Carfilzomib (crystalline oramorphous); Hamilton Syringe Gastight, 22 gauge; Dialysis kit,Spectra/Por® Float-A-Lyzer® G2 (Spectrum Laboratories Inc., RanchoDominguez, Calif.) molecular weight cut off (MWCO) 8-10 kD; Water forInjection (WFI); and Phosphate buffer saline 1× (PBS).

A lipid/ethanol solution was prepared as follows: 2 mL of ethanolcontaining 125 mg/mL egg sphingomyelin, 31.25 mg/mL cholesterol, 12.5mg/mL mPEG-DSPE. If needed, the lipid/ethanol solution was sonicatedseveral minutes to facilitate dissolution.

An aqueous solution of CFZ was prepared as follows: 10 mL of a 0.1M HClaqueous solution was prepared (˜pH 1) and CFZ in excess of solubilitywas added. The aqueous solution was sonicated in heated water bath (˜30°C.) for 20-30 minutes. Approximate carfilzomib solubility at pH 1 is 1.8mg/mL. Undissolved excess drug was removed by filtering through a 0.2 μmfilter to yield a visibly clear solution.

Alternatively, a supersaturated solution of carfilzomib is prepared bydissolving amorphous carfilzomib in 0.1M HCl solution with 6% (v/v)ethanol as a cosolvent followed by sonication in a warm water bath untilsolution becomes clear.

Liposomes were formed by rapid injection of 1 mL of the lipid-ethanolsolution into 9 mL of the aqueous solution of CFZ with stirring using amagnetic stir bar. Stirring was continued for 5-10 minutes. The solutionpH was ˜pH 1. The resulting solution was dialyzed against phosphatebuffer saline (or WFI) using the Dialysis kit (Spectra/Por®Float-A-Lyzer® G2) MWCO 8-10 kD for 12 to 16 hours. The bulk dialysissolution was replaced with fresh PBS or WFI after approximately 6-8hours. Solution pH of the dialyzed liposome containing formulation wasabout pH 3 to 3.5. The pH of the aqueous solution comprising theliposomes was adjusted with sodium hydroxide to between pH 3.5 to aphysiologic pH, ˜pH 6.8.

Table 12 presents nominal concentrations of the components of exemplaryliposomal compositions.

TABLE 12 Composition Drug Designation Lipid composition content * apL-1525 mg/mL egg SPH, 6.3 mg/mL cholesterol, 0.6 mg/mL 2.5 mg/mL mPEG-DSPEapL-11b 12.5 mg/mL EPC, 3.1 mg/mL cholesterol,   1 mg/mL 1.2 mg/mLmPEG-DSPE * Total drug content may include unencapsulated drug that wasnot removed during processing.

Example 11 Liposomes Comprising Entrapped Peptide Epoxyketone InduceAnti-Tumor Response

To evaluate the anti-cancer effect of liposomal compositions comprisingliposomes comprising peptide epoxyketone compounds, an exemplaryliposomal composition was tested in a mouse xenograft tumor model.

The liposomal composition was made by the methods described in Example 1and Example 2. The liposomal composition was as follows: pL6=2 mg/mLCFZ, 12.5 mg/mL Sphingomylin, 3.2 mg/mL cholesterol, 1.3 mg/mLmPEG-DSPE.

First, the pharmacodynamic response of injectable carfilzomib formulatedin SBE-CD (non-liposomal; see, e.g., U.S. Patent Publication Nos.2011/0236428) and the pL-6 a liposomal composition comprising liposomesloaded with CFZ was evaluated in BALB/C mice following a singleintravenous bolus administration.

The mice (three mice per time point) were administered a dose of 10mg/kg carfilzomib formulated in SBE-CD (non-liposomal) or 15 mg/kg ofpL-6 a liposomal composition comprising liposomes loaded with CFZ as asolution in a dose volume of 5 mL/kg. Blood samples and tissues forpharmacodynamic testing were taken at 0, 1, 4, 6 and 24 hours afteradministration of the liposomal formulation and 0, 1, 8, and 24 hoursfor the non-liposomal formulation. Three tissue samples were evaluatedper time point for each tissue from each mouse. The pharmacodynamicresponse was determined by measurement of proteasome activity in wholeblood (primarily erythrocytes), adrenal, liver, and heart using afluorogenic substrate (LLVY-AMC; as described by Lightcap E S, McCormackT A, Pien C S, et al., Clin. Chem. 46:673-683 (2000)) to quantitate thechymotrypsin-like activity of the proteasome. All samples werenormalized to the corresponding vehicle without CFZ, and the vehiclesample time point measurement was 1 hour post dose.

A single dose of injectable carfilzomib formulated in SBE-B-CD(non-liposomal) at 10 mg/kg (MTD) or liposomal compositions comprisingliposomes comprising entrapped CFZ (pL-6) at 15 mg/kg resulted in arapid inhibition of >80% of proteasome activity within 1 hour in wholeblood and all other tissues: whole blood (primarily erythrocytes) (seeFIG. 5A), heart (see FIG. 5B), liver (see FIG. 5C), and adrenal (seeFIG. 5D). Similar and near complete recovery from proteasome inhibitionwas observed 24 hours post-dose in all tissues tested except for theblood and heart and occurred with a t1/2 of 6-24 hours for thenon-liposomal injectable CFZ. Delayed recovery of proteasome activity inthe adrenals was observed with the liposomal composition pL-6 suggestinglong-term exposure of CFZ. As expected, there was no recovery ofproteasome activity in blood due to the irreversible binding ofcarfilzomib and the lack of the erythrocytes to synthesize newproteasome.

These observations indicate that inhibition of proteasome activity inwhole blood and tissues is rapid and similar across compositions. Thedelay in recovery of proteasome activity in the adrenals suggestsextended exposure with the liposomal composition. The liposomalcompositions did not adversely affect biodistribution of CFZ.

Second, circulation half-life of liposomal CFZ was evaluated in 7-8 weekold female BALB/c mice (3/timepoint) following a single i.v. injectionof the following: injectable carfilzomib formulated in SBE-CD(non-liposomal) administered at 5 mg/kg; and the pL-6 a liposomalcomposition comprising liposomes loaded with CFZ administered at 15mg/kg.

As shown in FIG. 6, the plasma concentration of injectable carfilzomibSBE-CD composition (non-liposomal) declined rapidly followingintravenous, bolus administration and was below the limit ofquantitation after 1 hour. The half-life was about 20 minutes. This isdue to rapid and extensive metabolism (FIG. 6, line with open circles).

When delivered in the pL-6 liposomal composition, the duration ofexposure to carfilzomib was greatly extended (FIG. 6, line with solidsquares). Plasma concentration of total drug (encapsulated and released)declined slowly and was detectable for up to 24 hours post-dose. This isconsistent with the observed delay in proteasome recovery in tissues.

The data for the liposomal CFZ compositions versus CFZ SBE-CD(non-liposomal) demonstrate the ability of liposomal compositions tosignificantly enhance the circulation half-life of peptide epoxyketonecompounds. Further, the data show the ability to provide extendedduration of exposure to peptide epoxyketone compounds in liposomalcompositions versus the non-liposomal SBE-CD composition.

Third, the anti-tumor response of injectable carfilzomib formulated inSBE-CD (non-liposomal) and the pL-6 liposomal composition comprisingliposomes loaded with CFZ was evaluated in mice. Tumors were establishedby s.c. injection of RL cells (human non-Hodgkin's B cell lymphomacells; passage number <9 and viability >95% at the time of implantation)in the right flank of BNX mice (n=8-10 per group). For RL studies, cellsuspensions containing 1×10⁷ cells in a volume of 0.1 mL were injected.Mice were randomized into treatment groups and dosing initiated whentumors reached ˜100 mm³ (RL). Tumors were measured thrice weekly byrecording the longest perpendicular diameters and tumor volumes werecalculated using the equation V (in mm³)=(length×width)/2.

BNX mice bearing established human tumor xenograft derived from RL cellswere treated with either non-liposomal carfilzomib or liposomalcarfilzomib. Drug was administered on either a weekly (QW) schedule or aschedule of two consecutive daily doses administered each week (QD×2).The group sizes were N=8-10 mice/group.

The results are presented in FIG. 7. The data presented in the figuredemonstrate that weekly IV administration of liposomal compositionscomprising carfilzomib (FIG. 7, liposomal composition 15 mg/kg, QW, opentriangles) and QD×2 administration of liposomal compositions (FIG. 7,liposomal composition, 10 mg/kg CFZ, QD×2, solid circles) inducedanti-tumor responses similar to injectable carfilzomib formulated inSBE-CD (non-liposomal; FIG. 7, QD×2, 5 mg/kg) administered on a Day1/Day 2 schedule (i.e., QD×2). Statistical comparisons between treatmentgroups and vehicle controls were made by one-way ANOVA and Bonferronipost-hoc analysis (significance was p<0.001). The data show that theliposomal composition administered at 15 mg/kg once a week was asefficacious as a liposomal or non-liposomal composition administeredQD×2.

These data demonstrate that liposomal compositions comprising peptideepoxyketone compounds maintain efficacy at a reduced dosing frequencyrelative to a non-liposomal formulation.

As is apparent to one of skill in the art, various modification andvariations of the above embodiments can be made without departing fromthe spirit and scope of this invention. Such modifications andvariations are within the scope of this invention.

What is claimed is:
 1. A pharmaceutical liposomal compositioncomprising: liposome entrapped peptide epoxyketone compound, wherein (i)liposomes of the liposomal composition comprise one or more lipids, thelipids comprising at least one phospholipid selected from the groupconsisting of L-α-phosphatidylcholine,1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and sphingomyelin; (ii)the liposomes comprising a weight ratio of peptide epoxyketonecompound:lipid of between about 0.01:1 and about 1:1; and (iii) theliposomes have an average size of between about 0.05 microns and about0.5 microns; and an aqueous solution comprising the liposomes.
 2. Thepharmaceutical liposomal composition of claim 1, wherein the weightratio of peptide epoxyketone compound:lipid is between about 0.05:1 andabout 0.5:1.
 3. The pharmaceutical liposomal composition of anypreceding claim, wherein the liposomes have an average size of betweenabout 0.05 microns and about 0.2 microns.
 4. The pharmaceuticalliposomal composition of any preceding claim, wherein the lipids of theliposomes comprise between about 20 and about 100 weight percentphospholipid.
 5. The pharmaceutical liposomal composition of anypreceding claim, wherein the lipids of the liposomes further comprisebetween about 10 and about 50 weight percent cholesterol.
 6. Thepharmaceutical liposomal composition of any preceding claim, wherein thelipids of the liposomes further comprise between about 1 and about 20weight percent of a hydrophilic polymer-derivatized lipid.
 7. Thepharmaceutical liposomal composition of claim 6, wherein the hydrophilicpolymer of the hydrophilic polymer-derivatized lipid is a polyethyleneglycol.
 8. The pharmaceutical liposomal composition of claim 6 or claim7, wherein the lipid of the hydrophilic polymer-derivatized lipid is acholesterol or a phospholipid.
 9. The pharmaceutical liposomalcomposition of claim 8, wherein the hydrophilic polymer-derivatizedlipid is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000].
 10. The pharmaceutical liposomal composition of anypreceding claim, wherein the lipids of the liposomes compriseL-α-phosphatidylcholine.
 11. The pharmaceutical liposomal composition ofany preceding claim, wherein the lipids of the liposomes comprisesphingomyelin.
 12. The pharmaceutical liposomal composition of anypreceding claim, wherein the lipids of the liposomes comprise1,2-distearoyl-sn-glycero-3-phosphocholine.
 13. The pharmaceuticalliposomal composition of any preceding claim, wherein the lipids of theliposomes comprise 1,2-dipalmitoyl-sn-glycero-3-phosphocholine.
 14. Thepharmaceutical liposomal composition of any preceding claim, wherein thelipids of the liposomes comprise1,2-distearoyl-sn-glycero-3-phosphoethanolamine.
 15. The pharmaceuticalliposomal composition of any preceding claim, wherein the lipids of theliposomes further comprise about 0.001 to about 5 weight percentα-tocopherol.
 16. The pharmaceutical liposomal composition of anypreceding claim, wherein the aqueous solution further comprises abuffer.
 17. The pharmaceutical liposomal composition of any precedingclaim, wherein the aqueous solution further comprises an agent tomaintain isotonicity.
 18. The pharmaceutical liposomal composition ofany preceding claim, wherein the liposomal composition comprisesliposomes comprising the peptide epoxyketone compound and a solubilizingagent in an internal aqueous core of the liposomes.
 19. Thepharmaceutical liposomal composition of claim 18, wherein thesolubilizing agent, is a compound.
 20. The pharmaceutical liposomalcomposition of claim 19, wherein the solubilizing agent is a compound,the compound is a cyclodextrin, and the liposomes of the liposomalcomposition comprise the peptide epoxyketone compound complexed with thecyclodextrin in the internal aqueous core of the liposomes.
 21. Thepharmaceutical liposomal composition of claim 20, wherein thecyclodextrin is a sulfobutylether-betacyclodextrin or ahydroxypropyl-betacyclodextrin.
 22. The pharmaceutical liposomalcomposition of claim 21, wherein the cyclodextrin is asulfobutylether-betacyclodextrin.
 23. The pharmaceutical liposomalcomposition of claim 21, wherein the cyclodextrin is ahydroxypropyl-betacyclodextrin.
 24. The pharmaceutical liposomalcomposition of any preceding claim, wherein the aqueous solution ofliposomal composition is adjusted to between about pH 3.5 and about pH7.0.
 25. The pharmaceutical liposomal composition of claim 24, whereinthe aqueous solution of liposomal composition is adjusted to a humanphysiological pH.
 26. The pharmaceutical liposomal composition of anypreceding claim, wherein the peptide epoxyketone compound is selectedfrom the group consisting of compound II, compound III, compound IV, andcompound V.
 27. The pharmaceutical liposomal composition of anypreceding claim, wherein the peptide epoxyketone compound is carfilzomib(compound V).
 28. A dry pharmaceutical composition formed by drying thepharmaceutical liposomal composition of any preceding claim.
 29. A drypharmaceutical composition comprising: one or more lipids, the lipidscomprising at least one phospholipid selected from the group consistingof L-α-phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and sphingomyelin; anda weight ratio of peptide epoxyketone compound:lipid of between about0.01:1 and about 1:1.
 30. The dry pharmaceutical composition of claim29, further comprising additional excipients.
 31. The dry pharmaceuticalformulation of claim 30, wherein the additional excipients comprise oneor more excipient selected from the group consisting of a cryoprotectantagent, a sugar, a glass transition modifying agent, and combinationsthereof.
 32. A method of making a pharmaceutical liposomal compositioncomprising: preparing a dried film comprising (i) a peptide epoxyketonecompound and one or more lipids, the lipids comprising at least onephospholipid selected from the group consisting ofL-α-phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and sphingomyelin, and(ii) a weight ratio of peptide epoxyketone compound:lipid of betweenabout 0.01:1 and about 1:1; and rehydrating the dried film with anaqueous solution to form a liposomal composition comprising liposomesand the aqueous solution.
 33. The method of claim 32, wherein the weightratio of peptide epoxyketone compound:lipid is between about 0.05:1 andabout 0.5:1.
 34. The method of claim 32 or claim 33, further comprisingsizing the liposomes to have an average size of between about 0.05microns and about 0.5 microns.
 35. The method of claim 34, comprisingsizing the liposomes to have an average size of between about 0.05microns and about 0.2 microns.
 36. The method of any one of claims32-35, wherein the lipids of the dried film comprise between about 20and about 100 weight percent phospholipid.
 37. The method of any ofclaims 32-36, wherein the lipids of the dried film further comprisebetween about 10 and about 50 weight percent cholesterol.
 38. The methodof any of claims 32-37, wherein the lipids of the dried film furthercomprise between about 1 and about 20 weight percent of a hydrophilicpolymer-derivatized lipid.
 39. The method of claim 38, wherein thehydrophilic polymer of the hydrophilic polymer-derivatized lipid is apolyethylene glycol.
 40. The method of claim 38 or claim 39, wherein thelipid of the hydrophilic polymer-derivatized lipid is a cholesterol or aphospholipid.
 41. The method of claim 40, wherein the hydrophilicpolymer-derivatized lipid is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000].
 42. The method of any of claims 32-41, wherein the lipidsof the dried film comprise L-α-phosphatidylcholine.
 43. The method ofany of claims 32-42, wherein the lipids of the dried film comprisesphingomyelin.
 44. The method of any of claims 32-43, wherein the lipidsof the dried film comprise 1,2-distearoyl-sn-glycero-3-phosphocholine.45. The method of any of claims 32-44, wherein the lipids of the driedfilm comprise 1,2-dipalmitoyl-sn-glycero-3-phosphocholine.
 46. Themethod of any of claims 32-45, wherein the lipids of the dried filmcomprise 1,2-distearoyl-sn-glycero-3-phosphoethanolamine.
 47. The methodof any of claims 32-46, wherein the lipids of the dried film furthercomprise about 0.001 to about 5 weight percent α-tocopherol.
 48. Themethod of any of claims 32-47, further comprising adding a buffer to theaqueous solution.
 49. The method of any of claims 32-48, furthercomprising adding an agent to maintain isotonicity to the aqueoussolution.
 50. The method of any of claims 32-49, further comprisingadjusting the pH of the aqueous solution to between about pH 3.5 andabout pH 7.0.
 51. The method of any of claims 32-49, further comprisingadjusting the pH of the aqueous solution to a human physiological pH.52. The method of any of claims 32-51, wherein the peptide epoxyketonecompound is selected from the group consisting of compound II, compoundIII, compound IV, and compound V.
 53. The method of any of claims 32-50,wherein the peptide epoxyketone compound is carfilzomib (compound V).54. A pharmaceutical liposomal composition made by the method of any ofclaims 32-53.
 55. A method of making a pharmaceutical liposomalcomposition comprising: preparing a dried film comprising one or morelipids, the lipids comprising at least one phospholipid selected fromthe group consisting of L-α-phosphatidylcholine,1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and sphingomyelin; andrehydrating the dried film with an aqueous solution comprising a peptideepoxyketone compound and one or more solubilizing agent to form aliposomal composition comprising liposomes dispersed in the aqueoussolution.
 56. The method of claim 55, wherein the one or moresolubilizing agent, is selected from the group consisting of a compound,a pH adjusting agent, a cosolvent, and a combination thereof.
 57. Themethod of claim 56, wherein the one or more solubilizing agent comprisesa compound and the compound is a cyclodextrin.
 58. The method of claim57, wherein the cyclodextrin is a sulfobutylether-betacyclodextrin or ahydroxypropyl-betacyclodextrin.
 59. The method of any of claims 56-58,wherein the one or more solubilizing agent comprises a pH adjustingagent and the aqueous solution has a pH of between about pH 0.5 andabout pH 3.0.
 60. The method of claim 59, wherein the pH of the aqueoussolution is about pH 1.0.
 61. The method of any of claims 56-60, whereinthe one or more solubilizing agent comprises a cosolvent.
 62. The methodof any of claims 55-61, further comprising dialysis, desalting, bufferexchange, or gel filtration.
 63. The method of any of claims 55-62,wherein the liposomes of the liposomal composition comprise a weightratio of peptide epoxyketone compound:lipid of between about 0.01:1 andabout 1:1.
 64. The method of any of claims 55-63, further comprisingsizing the liposomes to have an average size of between about 0.05microns and about 0.5 microns.
 65. The method of claim 64, comprisingsizing the liposomes to have an average size of between about 0.05microns and about 0.2 microns.
 66. The method of any one of claims55-65, wherein the lipids of the dried film comprise between about 20and about 100 weight percent phospholipid.
 67. The method of any ofclaims 55-66, wherein the lipids of the dried film further comprisebetween about 10 and about 50 weight percent cholesterol.
 68. The methodof any of claims 55-67, wherein the lipids of the dried film furthercomprise between about 1 and about 20 weight percent of a hydrophilicpolymer-derivatized lipid.
 69. The method of claim 68, wherein thehydrophilic polymer of the hydrophilic polymer-derivatized lipid is apolyethylene glycol.
 70. The method of claim 68 or claim 69, wherein thelipid of the hydrophilic polymer-derivatized lipid is a cholesterol or aphospholipid.
 71. The method of claim 70, wherein the hydrophilicpolymer-derivatized lipid is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000].
 72. The method of any of claims 55-71, wherein the lipidsof the dried film comprise L-α-phosphatidylcholine.
 73. The method ofany of claims 55-72, wherein the lipids of the dried film comprisesphingomyelin.
 74. The method of any of claims 55-73, wherein the lipidsof the dried film comprise 1,2-distearoyl-sn-glycero-3-phosphocholine.75. The method of any of claims 55-74, wherein the lipids of the driedfilm comprise 1,2-dipalmitoyl-sn-glycero-3-phosphocholine.
 76. Themethod of any of claims 55-75, wherein the lipids of the dried filmcomprise 1,2-distearoyl-sn-glycero-3-phosphoethanolamine.
 77. The methodof any of claims 55-76, wherein the lipids of the dried film furthercomprise about 0.001 to about 5 weight percent α-tocopherol.
 78. Themethod of any of claims 55-77, further comprising, after forming theliposomal composition, removing peptide epoxyketone compound fromnon-encapsulated aqueous solution.
 79. The method of claim 78, whereindialysis, ultracentrifugation, or gel filtration is used for removingthe peptide epoxyketone compound from the non-encapsulated aqueoussolution.
 80. The method of any of claims 55-79, further comprising,after forming the liposomal composition, adjusting the aqueous solutionto a pH of between about pH 3.5 and about pH 7.0.
 81. The method of anyof claims 55-79, further comprising, after forming the liposomalcomposition, adjusting the aqueous solution to a human physiological pH.82. The method of any of claims 55-81, further comprising, after formingthe liposomal composition, adding an agent to the aqueous solution tomaintain isotonicity.
 83. The method of any of claims 55-82, wherein thepeptide epoxyketone compound is selected from the group consisting ofcompound II, compound III, compound IV, and compound V.
 84. The methodof any of claims 55-83, wherein the peptide epoxyketone compound iscarfilzomib (compound V).
 85. A pharmaceutical liposomal compositionmade by the method of any of claims 55-84.
 86. A method of making apharmaceutical liposomal composition comprising: preparing a lipidsolution comprising a solvent and one or more lipids, the lipidscomprising at least one phospholipid selected from the group consistingof L-α-phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and sphingomyelin; andinjecting the lipid solution into an aqueous solution comprising apeptide epoxyketone compound and one or more solubilizing agent to forma liposomal composition comprising liposomes dispersed in the aqueoussolution.
 87. The method of claim 86, wherein the solvent is an organicsolvent.
 88. The method of claim 87, wherein the solvent is ethanol. 89.The method of any of claims 86-88, wherein the one or more solubilizingagent is a compound, a pH adjusting agent, a cosolvent, or a combinationthereof.
 90. The method of claim 89, wherein the one or moresolubilizing agent comprises a compound and the compound is acyclodextrin.
 91. The method of claim 90, wherein the cyclodextrin is asulfobutylether-betacyclodextrin or a hydroxypropyl-betacyclodextrin.92. The method of any of claims 89-91, wherein the one or moresolubilizing agent comprises a pH adjusting agent and the aqueoussolution has a pH of between about pH 0.5 and about pH 3.0.
 93. Themethod of claim 92, wherein the pH of the aqueous solution is about pH1.0.
 94. The method of any of claims 89-93, wherein the solubilizingagent comprises a cosolvent.
 95. The method of any of claims 86-94,further comprising dialysis, desalting, buffer exchange, or gelfiltration.
 96. The method of any of claims 86-95, wherein the liposomesof the liposomal composition comprise a weight ratio of peptideepoxyketone compound:lipid of between about 0.01:1 and about 1:1. 97.The method of any of claims 86-96, further comprising sizing theliposomes to have an average size of between about 0.05 microns andabout 0.5 microns.
 98. The method of claim 97, comprising sizing theliposomes to have an average size of between about 0.05 microns andabout 0.2 microns.
 99. The method of any one of claims 86-98, whereinthe lipids of the liposomes of the liposomal composition comprisebetween about 20 and about 100 weight percent phospholipid.
 100. Themethod of any of claims 86-99, wherein the lipids of the liposomes ofthe liposomal composition further comprise between about 10 and about 50weight percent cholesterol.
 101. The method of any of claims 86-100,wherein the lipids of the liposomes of the liposomal composition furthercomprise between about 1 and about 20 weight percent of a hydrophilicpolymer-derivatized lipid.
 102. The method of claim 101, wherein thehydrophilic polymer of the hydrophilic polymer-derivatized lipid is apolyethylene glycol.
 103. The method of claim 101 or claim 102, whereinthe lipid of the hydrophilic polymer-derivatized lipid is a cholesterolor a phospholipid.
 104. The method of claim 103, wherein the hydrophilicpolymer-derivatized lipid is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000].
 105. The method of any of claims 86-104, wherein thelipids of the liposomes of the liposomal composition compriseL-α-phosphatidylcholine.
 106. The method of any of claims 86-105,wherein the lipids of the liposomes of the liposomal compositioncomprise sphingomyelin.
 107. The method of any of claims 86-106, whereinthe lipids of the liposomes of the liposomal composition comprise1,2-distearoyl-sn-glycero-3-phosphocholine.
 108. The method of any ofclaims 86-107, wherein the lipids of the liposomes of the liposomalcomposition comprise 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. 109.The method of any of claims 86-108, wherein the lipids of the liposomesof the liposomal composition comprise1,2-distearoyl-sn-glycero-3-phosphoethanolamine.
 110. The method of anyof claims 86-109, wherein the lipids of the liposomes of the liposomalcomposition further comprise about 0.001 to about 5 weight percentα-tocopherol.
 111. The method of any of claims 86-110, furthercomprising, after forming the liposomal composition, removing peptideepoxyketone compound from non-encapsulated aqueous solution.
 112. Themethod of claim 111, wherein dialysis, ultracentrifugation, or gelfiltration is used for removing peptide epoxyketone compound from thenon-encapsulated aqueous solution.
 113. The method of any of claims86-112, further comprising, after forming the liposomal composition,adjusting the aqueous solution to a pH of between about pH 3.5 and aboutpH 7.0.
 114. The method of any of claims 86-112, further comprising,after forming the liposomal composition, adjusting the aqueous solutionto a human physiological pH.
 115. The method of any of claims 86-114,further comprising, after forming the liposomal composition, adding anagent to the aqueous solution to maintain isotonicity.
 116. The methodof any of claims 86-115, wherein the peptide epoxyketone compound isselected from the group consisting of compound II, compound III,compound IV, and compound V.
 117. The method of any of claims 86-116,wherein the peptide epoxyketone compound is carfilzomib (compound V).118. A pharmaceutical liposomal composition made by the method of any ofclaims 86-117.
 119. A method of treating multiple myeloma in a subjectin need of treatment, comprising: administering a therapeuticallyeffective amount of a pharmaceutical liposomal composition any one ofclaim 1-27, 54, 85, or
 118. 120. The method of claim 119, furthercomprising simultaneous, sequential, or separate administration of atherapeutically effective amount of a chemotherapeutic agent.
 121. Amethod of treating a solid tumor in a subject in need of treatment,comprising: administering a therapeutically effective amount of apharmaceutical liposomal composition any one of claim 1-27, 54, 85, or118.
 122. The method of claim 121, further comprising simultaneous,sequential, or separate administration of a therapeutically effectiveamount of a chemotherapeutic agent.
 123. A method of treating a diseaseor condition in a subject in need of treatment, comprising:administering a therapeutically effective amount of a pharmaceuticalliposomal composition any one of claim 1-27, 54, 85, or
 118. 124. Themethod of claim 123, further comprising simultaneous, sequential, orseparate administration of a therapeutically effective amount of achemotherapeutic agent, a cytokine, a steroid, or an immunotherapeuticagent.